Aldolase and production process of substituted α-keto acids

ABSTRACT

4-(Indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate, which is useful as an intermediate in the synthesis of monatin, may be synthesized from indole pyruvic acid and pyruvic acid (and/or oxaloacetic acid) by using a novel aldolase derived from the genus  Pseudomonas, Erwinia, Flavobacterium , or  Xanthomonas.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is divisional of U.S. Ser. No. 11/960,116 (nowU.S. Pat. No. 8,058,038), filed Dec. 19, 2007, which is a divisional ofU.S. Ser. No. 11/066,542 (now U.S. Pat. No. 7,351,569), filed on Feb.28, 2005, which is a continuation of PCT/JP03/10750, filed on Aug. 26,2003, and claims priority to JP 2002-245980, filed on Aug. 26, 2002, andJP 2003-171299, filed on Jun. 16, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an aldolase and a process for producingsubstituted α-keto acid, and more particularly, to an aldolase that maybe preferably used in the synthesis of4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (hereinafter, “IHOG”),which is useful as an intermediate in monatin synthesis, and a processfor producing substituted α-keto acids.

2. Discussion of the Background

Monatin, which has the structure shown in formula (5) below, a naturallysweet amino acid that is isolated and extracted from the roots of shrubsin Southern Africa. It has a potent sweet taste equivalent to severalten to several thousand times that of sucrose, and is expected to beuseful as a sweetener. However, the usefulness of monatin has only beenrecently discovered, and a process for synthesizing monatin as the levelof industrial production has yet to be established.

Under these circumstances, the inventors of the present inventiondeveloped a novel process for producing monatin composed of thefollowing reactions (1) and (2) by using indole pyruvic acid and pyruvicacid which may be acquired as reagents.

(1) A reaction step of synthesizing precursor keto acid (IHOG) by aldolcondensation of indole pyruvic acid and pyruvic acid (and/or oxaloaceticacid); and,

(2) a reaction step of aminating the second position of IHOG.

There have been no previous reports of examples of synthesizingprecursor keto acid (IHOG) from indole pyruvic acid and pyruvic acid (oroxaloacetic acid) using a microbial enzyme system for the aldolcondensation reaction of (1) in the aforementioned synthesis route ofmonatin.

Examples of microbial enzymes that catalyze aldol condensation using twomolecules of α-keto acid (or substituted α-keto acid) as a substratereported thus far include 4-hydroxy-4-methyl-2-oxoglutarate aldolasederived from bacteria belonging to the genus Pseudomonas, and4-hydroxy-2-oxoglutarate aldolase present in E. coli, B. subtilis, andso forth.

The former 4-hydroxy-4-methyl-2-oxoglutarate aldolase has been reportedto catalyze a reaction in which 4-hydroxy-4-methyl-2-oxoglutarate(4-HMG) is formed from two molecules of pyruvic acid, and a reaction inwhich one molecule of oxaloacetic acid and one molecule of pyruvic acidare formed from 4-oxalocitramalate (Kiyofumi Maruyama, Journal ofBiochemistry, 108, 327-333 (1990)). Furthermore, the latter4-hydroxy-2-oxoglutarate aldolase is known to catalyze a reaction inwhich 4-hydroxy-2-oxoglutarate (4HG) is formed from one molecule ofglyoxylic acid and one molecule of pyruvic acid.

However, there have been no reports or findings indicating that any ofthese microorganisms are associated with activity that cleaves4-phenylmethyl-4-hydroxy-2-oxoglutarate (PHOG) or activity thatsynthesizes a precursor keto acid (IHOG) of monatin from indole pyruvicacid and pyruvic acid (or oxaloacetic acid), and whether or not thealdolases produced by these microbial strains may be used in theaforementioned synthesis route of monatin is unknown.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an aldolase that maybe appropriately used in the synthesis of IHOG, which is useful as anintermediate in the synthesis of monatin, and a process for producingsubstituted α-keto acids.

As a result of extensive research conducted in consideration of theaforementioned problems, the inventors of the present invention foundthat an aldolase that may be preferably used in the synthesis of thedesired IHOG is present in certain microbial species, thereby leading tocompletion of the present invention.

Namely, the present invention is as described below.

[1] A DNA of following (a) or (b):

(a) A DNA comprising the nucleotide sequence of SEQ ID No: 1 or thenucleotide sequence of base numbers 444 to 1118 or base numbers 456 to1118 of the same sequence;

(b) A DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 1 or a nucleotide sequence of base numbers 444 to 1118 or 456 to1118 of the same sequence, and encodes a protein having aldolaseactivity.[2] A DNA of following (c) or (d):(c) A DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 2 or an amino acid sequence of residue numbers 5 to 225 ofthe same sequence;(d) A DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 2 or an amino acid sequence of residue numbers 5 to 225 of the samesequence, and has aldolase activity.[3] A DNA of following (e) or (f):(e) A DNA comprising the nucleotide sequence of SEQ ID No: 15 or anucleotide sequence of base numbers 398 to 1141 of the same sequence;(f) A DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 15 or a nucleotide sequence of base numbers 398 to 1141 of the samesequence, and encodes a protein having aldolase activity.[4] A DNA of following (g) or (h):(g) A DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 16;(h) A DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 16, and has aldolase activity.[5] A recombinant DNA obtainable from ligating the DNA according to anyof [1] to [4] with a vector DNA.[6] A cell transformed by the recombinant DNA according to [5].[7] A process for producing a protein having aldolase activitycomprising: cultivating cells according to [6] in a medium, andaccumulating a protein having aldolase activity in the any one of mediumand cells or both.[8] A protein of following (i) or (j):(i) a protein comprising the amino acid sequence of SEQ ID No: 2 or anamino acid sequence of residue numbers 5 to 225 of the same sequence;(j) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID No: 2or an amino acid sequence of residue numbers 5 to 225 of the samesequence, and has aldolase activity.[9] A protein of following (k) or (l):(k) a protein comprising the amino acid sequence of SEQ ID No: 16;(l) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID No: 16,and has aldolase activity.[10] A protein which:(A) has at least one of activities that catalyze reactions of:

-   4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate is formed by aldol    condensation of indole-3-pyruvic acid and pyruvic acid, and-   4-phenylmethyl-4-hydroxy-2-oxoglutarate is formed by aldol    condensation of phenyl pyruvic acid and pyruvic acid,    (B) the optimum pH of the activity according to (A) is about 9 at    33° C., and    (C) the molecular weight as measured by gel filtration is about 146    kDa, and the molecular weight per subunit as measured by SDS-PAGE is    about 25 kDa.    [11] A protein which:    (A) has aldolase activity,    (B) is derived from Pseudomonas species,    (C) has pH stability at pH 6 and above,    (D) has temperature stability at 70° C. or lower, and    (E) the molecular weight as measured by gel filtration is about 146    kDa, and the molecular weight per subunit as measured by SDS-PAGE is    about 25 kDa.    [12] A protein according to [11], wherein the aldolase activity is    improved by containing inorganic phosphate in the enzyme reaction    mixture.    [13] A composition having aldolase activity obtainable from    cultivating Pseudomonas species bacteria in a medium, accumulating    any of the proteins of following (i) to (l) in any one of the medium    and cells or both, and purifying any one of the medium and cells or    both:    (i) a protein comprising the amino acid sequence of SEQ ID No: 2 or    an amino acid sequence of residue numbers 5 to 225 of the same    sequence;    (j) a protein having an amino acid sequence that contains a    substitution, deletion, insertion, addition or inversion of one or    several amino acid residues in the amino acid sequence of SEQ ID No:    2 or an amino acid sequence of residue numbers 5 to 225 of the same    sequence, and has aldolase activity;    (k) a protein comprising the amino acid sequence of SEQ ID No: 16;    (l) a protein having an amino acid sequence that contains a    substitution, deletion, insertion, addition or inversion of one or    several amino acid residues in the amino acid sequence of SEQ ID No:    16, and has aldolase activity.    [14] A process for producing a substituted α-keto acid represented    by the following general formula (3):

(wherein R¹ represents a hydrogen atom, an alkyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms, acarboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof, and R¹ may be substituted with at least onesubstituent selected from the group consisting of a halogen atom, ahydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxygroup having up to 3 carbon atoms and an amino group; and R² representsa hydrogen atom, an alkyl group having 1 to 8 carbon atoms, an alkoxylgroup having 1 to 8 carbon atoms, a carboxyalkyl group having 2 to 9carbon atoms, an aryl or an aralkyl group having up to 20 carbon atoms,a heterocycle-containing hydrocarbon group having up to 11 carbon atoms,a hydroxyl group or an ester derivative thereof, and R² may besubstituted with at least one substituent selected from the groupconsisting of a halogen atom, a hydroxyl group, an alkyl group having upto 3 carbon atoms, an alkoxy group having up to 3 carbon atoms and anamino group; and when R¹ represents a hydrogen atom, a methyl group or acarboxymethyl group in general formula (1), R² does not represent ahydrogen atom) comprising a reaction of the substituted α-keto acidrepresented by the following general formula (1):

(wherein R¹ has the same meaning as R¹ in general formula (3)), with thesubstituted α-keto acid represented by the following general formula(2):

(wherein R² has the same meaning as defined in general formula (3));

wherein the reaction is performed in the presence of a protein thatcatalyzes the reaction.

[15] A process for producing a substituted α-keto acid according to[14], wherein R² represents a hydrogen atom or a carboxyl group.

[16] A process for producing a substituted α-keto acid according to[15], wherein R² represents a hydrogen atom.

[17] A process for producing a substituted α-keto acid according to[14], wherein R¹ represents a benzyl group or a 3-indolylmethyl group,and R² represents a hydrogen atom or a carboxyl group.

[18] A process for producing a substituted α-keto acid represented bythe following formula (4):

from oxaloacetic acid or pyruvic acid, and indole-3-pyruvic acid whereinthe reaction is performed in the presence of a protein that catalyzesthe reaction.[19] A process for producing a substituted α-keto acid according to [14]to [18], wherein the protein that catalyzes the reaction is derived froma microorganism selected from the group consisting of Pseudomonasspecies, Erwinia species, Flavobacterium species and Xanthomonasspecies.[20] A process for producing a substituted α-keto acid according to[19], wherein the microorganism is Pseudomonas taetrolens, Pseudomonascoronafaciens, Pseudomonas desmolytica, Erwinia sp., Flavobacteriumrhenanum or Xanthomonas citri.[21] A process for producing a substituted α-keto acid according to[20], wherein the microorganism is Pseudomonas taetrolens ATCC4683 orPseudomonas coronafaciens AJ2791.[22] A process for producing a substituted α-keto acid according to [14]to [21], wherein the protein that catalyzes the reaction is a proteinaccording to any of the following (i) to (l):(i) a protein comprising the amino acid sequence of SEQ ID No: 2 or anamino acid sequence of residue numbers 5 to 225 of the same sequence;(j) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition, or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID No: 2or an amino acid sequence of residue numbers 5 to 225 of the samesequence, and has aldolase activity;(k) a protein comprising the amino acid sequence of SEQ ID No: 16;(l) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition, or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID No: 16,and has aldolase activity.[23] A process for producing a substituted α-keto acid represented bythe following general formula (3′):

(wherein R¹ represents a hydrogen atom, an alkyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms, acarboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof, R³ represents a hydrogen atom or a carboxyl group,and R¹ may be substituted with at least one substituent selected fromthe group consisting of a halogen atom, a hydroxyl group, an alkyl grouphaving up to 3 carbon atoms, an alkoxy group having up to 3 carbonatoms, and an amino group; and R⁴ represents a hydrogen atom, an alkylgroup having 1 to 8 carbon atoms, an alkoxyl group having 1 to 8 carbonatoms, a carboxyalkyl group having 2 to 9 carbon atoms, an aryl or anaralkyl group having up to 20 carbon atoms, a heterocycle-containinghydrocarbon group having up to 11 carbon atoms, a hydroxyl group or anester derivative thereof, and R⁴ may be substituted with at least onetype of substituent selected from the group consisting of a halogenatom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, analkoxy group having up to 3 carbon atoms, and an amino group) comprisinga reaction of the compound represented by the following general formula(1′):

(wherein R¹ and R³ have the same meanings as defined in general formula(3′)), with the substituted α-keto acid represented by the followinggeneral formula (2′):

(wherein R⁴ has the same meaning as defined in general formula (3′));

wherein the reaction is performed in the presence of a protein accordingto [8] or [9].

[24] A process for producing a substituted α-keto acid according to[23], wherein R⁴ is a hydrogen atom or a carboxyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flowchart of the process for producing aldolase of thepresent invention.

FIG. 2 shows the results of measuring the aldolase activity of aldolasederived from Pseudomonas taetrolens ATCC4683 (PtALD) versus PHOGconcentration.

FIG. 3 shows the results of measuring the aldolase activity of PtALDversus MgCl₂ concentration.

FIG. 4 shows the results of measuring the aldolase activity of PtALDversus KPi concentration.

FIG. 5 shows the results of measuring the pH stability of PtALD.

FIG. 6 shows the results of measuring the temperature stability ofPtALD.

DETAILED DESCRIPTION OF THE INVENTION

The following provides a detailed explanation of [I] aldolase and [II] aprocess for producing substituted α-keto acid using aldolase of thepresent invention with reference to the accompanying drawings.

[I] Aldolase

According to research by the inventors of the present invention,bacterial strains were confirmed to exist that form aldolase having theability to cleave 4-phenylmethyl-4-hydroxy-2-oxoglutarate (PHOG) in thegenus Pseudomonas, Erwinia, Flavobacterium, and Xanthomonas.

Since the aldolase produced by these microorganisms catalyzes a reactionthat forms one molecule of phenyl pyruvic acid and one mole of pyruvicacid by cleaving one molecule of PHOG, the inventors of the presentinvention thought that the aldolase could catalyze a reaction in which4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG) is formed fromindole pyruvic acid and pyruvic acid (or oxaloacetic acid). On the basisof this approach, in order to clarify the existence of a novel aldolase,together with purifying and isolating aldolase from cultivated microbialcells of the microorganisms, the inventors of the present inventionfound that this enzyme synthesizes HOG by aldol condensation of indolepyruvic acid and pyruvic acid (or oxaloacetic acid).

Furthermore, the inventors of the present invention determined the aminoacid sequence of the aldolase by purifying aldolase derived fromPseudomonas taetrolens ATCC4638 (which may also be abbreviated asPtALD). Furthermore, the inventors of the present invention alsosynthesized a DNA molecule of about 30 base pairs deduced from the aminoacid sequence of the aldolase, isolated and obtained a fragment of DNAthat encodes the aldolase by PCR using this DNA molecule, and succeededin isolating the entire length of DNA that encodes PtALD from achromosome library of this microorganism by using the DNA fragment as aprobe.

The DNA that encodes the PtALD of the present invention that isidentified according to the aforementioned method is shown in SEQ ID No:1 in the Sequence Listing. Furthermore, the amino acid sequence of PtALDencoded by the nucleotide sequence of SEQ ID No: 1 is shown in SEQ IDNo: 2 and SEQ ID No: 3. SEQ ID No: 2 is the amino acid sequence of PtALDencoded by the nucleotide sequence of base numbers 456 to 1118 in thenucleotide sequence of SEQ ID No: 1. Furthermore, SEQ ID No: 3 is theamino acid sequence of PtALD encoded by the nucleotide sequence of basenumbers 444 to 1118 in the nucleotide sequence of SEQ ID No: 1, and isequivalent to the amino acid sequence of residue numbers 5 to 225 in theamino acid sequence of SEQ ID No: 2. Both of the PtALD of SEQ ID No: 2and SEQ ID No: 3 have aldolase activity, and catalyze the reaction inwhich 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG) shown in thefollowing formula (4) is synthesized from one molecule of indole pyruvicacid and one molecule of oxaloacetic acid (or pyruvic acid).

Furthermore, the inventors of the present invention also succeeded inisolating the entire length of DNA that encodes aldolase derived fromPseudomonas coronafaciens ATCC4683 (which may also be abbreviated asPcALD) from a chromosomal DNA library of the microorganism by using anobtained DNA fragment that encodes PtALD as a probe. A DNA that encodesthe PcALD of the present invention identified by the aforementionedmethod is shown in SEQ ID No: 15. The amino acid sequence of PcALDencoded by the nucleotide sequence of SEQ ID No: 15 is shown in SEQ IDNo: 16. SEQ ID No: 16 is an amino acid sequence of PcALD encoded by thenucleotide sequence of base numbers 398 to 1141 of the nucleotidesequence of SEQ ID No: 15. The PcALD of SEQ ID No: 16 also has aldolaseactivity and catalyzes the reaction in which4-(indol-3-ylmetyyl)-4-hydroxy-2-oxoglutarate (IHOG) shown in thefollowing formula (4) is synthesized from one molecule of indole pyruvicacid and one molecule of oxaloacetic acid (or pyruvic acid).

Next, a detailed explanation is provided of (1) a DNA encoding aldolase,(2) properties of aldolase and (3) a process for producing aldolase inthat order.

(1) A DNA Encoding Aldolase

The aldolase gene of the present invention having the nucleotidesequence of SEQ ID No: 1 was isolated from a chromosomal DNA ofPseudomonas taetrolens strain ATCC4683 as described above. Thenucleotide sequence of SEQ ID No: 1 demonstrates homology of 29% withknown 4-hydroxy-4-methyl-2-oxoglutarate aldolase (gene name: proA)(Biosci. Biotechnol. Biochem., 65(12), 2701-2709 (2001), Maruyama, K. etal.) derived from the bacterium, Pseudomonas ochraceae, at the aminoacid sequence level.

The aldolase gene of the present invention having the nucleotidesequence of SEQ ID No: 15 was isolated from a chromosomal DNA ofPseudomonas coronafaciens strain AJ2791 as described above. The aminoacid sequence encoded by the nucleotide sequence of SEQ ID No: 15demonstrates homology of 28% with known4-hydroxy-4-methyl-2-oxoglutarate aldolase (gene name: proA) (Biosci.Biotechnol. Biochem., 65(12), 2701-2709 (2001), Maruyama, K. et al.)derived from the bacterium, Pseudomonas ochraceae. Furthermore, theamino acid sequence encoded by the nucleotide sequence of SEQ ID No: 15demonstrates homology of 41% with the aldolase derived from the strainPseudomonas taetrolens ATCC 4683 described above.

Here, analysis of homology indicates the value obtained by calculatingthe parameter as the initial set value using the “Genetyx Ver. 6”genetic analytical software (Genetyx).

The following provides an explanation of the method for obtaining a DNAencoding aldolase from aldolase-producing bacteria.

First, the amino acid sequence of the purified aldolase is determined.At this time, the amino acid sequence may be determined by using themethod of Edman (Edman, P., Acta Chem. Scand. 4, 227 (1950)).Furthermore, the amino acid sequence may be determined using theSequencer made by Applied Biosystems. Following limited hydrolysis withprotease of the aldolase of the present invention derived fromPseudomonas taetrolens strain ATCC4683, peptide fragments werefractionated with reverse phase HPLC. Determination of the internalamino acid sequences of two of these fragments clearly revealed them tobe the sequences shown in SEQ ID No: 4 and SEQ ID No: 5.

The nucleotide sequence of a DNA that encodes these amino acid sequencesis then able to be deduced based on the amino acid sequences that aredetermined. Universal codons are employed to deduce the nucleotidesequence of the DNA.

DNA molecules of about 30 base pairs were then synthesized based on thededuced nucleotide sequence. The method used to synthesize the DNAmolecules is disclosed in Tetrahedron Letters, 22, 1859 (1981).Furthermore, the DNA molecules may also be synthesized using theSynthesizer made by Applied Biosystems. The DNA molecules may be used asa probe when isolating the entire length of DNA that encodes aldolasefrom a chromosomal DNA library of a microorganism that producesaldolase. Alternatively, they may also be used as a primer whenamplifying a DNA that encodes aldolase of the present invention by PCR.However, since the DNA amplified using PCR does not contain the entirelength of DNA that encodes aldolase, the entire length of DNA thatencodes aldolase is isolated from a chromosomal DNA library of amicroorganism that produces aldolase using the DNA amplified by PCR as aprobe.

The procedure employed for PCR is described in publications such asWhite, T. J. et al., Trends Genet. 5, 185 (1989). The method forisolating a chromosomal DNA, as well as the method for isolating adesired DNA molecule from a gene library using a DNA molecule as aprobe, are described in publications such as Molecular Cloning, 2ndedition, Cold Spring Harbor Press (1989).

A method for determining the nucleotide sequence of isolated a DNA thatencodes aldolase is described in A Practical Guide to Molecular Cloning,John Wiley & Sons, Inc. (1985). Furthermore, the nucleotide sequence maybe determined by using the DNA Sequencer made by Applied Biosystems. ADNA encoding aldolase derived from Pseudomonas taetrolens strainATCC4683 is shown in SEQ ID No: 1, while a DNA encoding aldolase derivedfrom Pseudomonas coronafaciens strain AJ2791 is shown in SEQ ID No: 15.

A DNA that encodes aldolase which catalyzes the reaction in which IHOGis formed from indole pyruvic acid and pyruvic acid (or oxaloaceticacid) is not only the DNA shown in SEQ ID Nos: 1 and 15. This is becausethere ought to be differences in nucleotide sequences observed for eachspecies and strain among Pseudomonas species that form aldolase whichcatalyzes the reaction in which IHOG is synthesized from indole pyruvicacid and pyruvic acid (or oxaloacetic acid).

The DNA of the present invention not only includes the isolated DNA thatencodes aldolase, but a DNA in which mutations have been artificiallyadded to a DNA that encodes aldolase isolated from a chromosomal DNA ofan aldolase-producing microorganism is also included in the DNA of thepresent invention when it encodes aldolase. Methods for artificiallyadding mutations include commonly used methods such as the method forintroducing site-specific mutations described in Method. in Enzymol.,154 (1987).

A DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 1, and encodes a protein having aldolase activity is also includedin the DNA of the present invention. As used herein, the “stringentconditions” refer to those conditions under which a specific hybrid isformed whereas an unspecific hybrid is not formed. Although it isdifficult to numerically express these conditions explicitly, by way ofexample, mention is made of those conditions under which DNA moleculeshaving higher homology e.g. preferably 50% or more, more preferably 80%or more, still more preferably 90% or more, and particularly preferably95% or more homology, hybridize with each other, while DNA moleculeshaving lower homology do not hybridize with each other, or thoseconditions under which hybridization occurs under usual washingconditions in Southern hybridization, that is, at a salt concentrationcorresponding to 0.1×SSC and 0.1% SDS at 37° C., preferably 0.1×SSC and0.1% SDS at 60° C., and more preferably 0.1×SSC and 0.1% SDS at 65° C.Furthermore, “aldolase activity” may be sufficient for the activity thatsynthesizes IHOG from indole pyruvic acid and pyruvic acid (oroxaloacetic acid). However, in the case of a nucleotide sequence thathybridizes under stringent conditions with a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID No: 1, it preferablyretains aldolase activity of 10% or more, preferably 30% or more, morepreferably 50% or more, and still more preferably 70% or more, ofprotein having the amino acid sequence of SEQ ID No: 2 or 3 underconditions of 33° C. and pH 9.

A DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 15, and encodes a protein having aldolase activity is also includedin the DNA of the present invention. As used herein, the “stringentconditions” refer to those conditions under which a specific hybrid isformed whereas an unspecific hybrid is not formed. Although it isdifficult to numerically express these conditions explicitly, by way ofexample, mention is made of those conditions under which DNA moleculeshaving higher homology e.g. preferably 50% or more, more preferably 80%or more, still more preferably 90% or more, and particularly preferably95% or more homology, hybridize with each other, while DNA moleculeshaving lower homology do not hybridize with each other, or thoseconditions under which hybridization occurs under usual washingconditions in Southern hybridization, that is, at a salt concentrationcorresponding to 0.1×SSC and 0.1% SDS at 37° C., preferably 0.1×SSC and0.1% SDS at 60° C., and more preferably 0.1×SSC and 0.1% SDS at 65° C.Furthermore, “aldolase activity” may be sufficient for the activity thatsynthesizes IHOG from indole pyruvic acid and pyruvic acid (oroxaloacetic acid). However, in the case of a nucleotide sequence thathybridizes under stringent conditions with a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID No: 15, it preferablyretains aldolase activity of 10% or more, preferably 30% or more, morepreferably 50% or more, and still more preferably 70% or more, ofprotein having the amino acid sequence of SEQ ID No: 16 under conditionsof 33° C. and pH 9.

Furthermore, a DNA that encodes a protein which is substantiallyidentical to the aldolase encoded by the DNA of SEQ ID No: 1 or 15 isalso included in the DNA of the present invention. Namely, the followingDNAs are also included in the DNA of the present invention:

(a) a DNA comprising the nucleotide sequence of base numbers 444 to 1118or 456-1118 in the nucleotide sequence of SEQ ID No: 1;

(b) a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of basenumbers 444 to 1118 or 456 to 1118 in the nucleotide sequence of SEQ IDNo: 1, and encodes a protein having aldolase activity;(c) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 2 or an amino acid sequence of residue numbers 5 to 225 ofthe same sequence;(d) a DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 2 or an amino acid sequence of residue numbers 5 to 225 of the samesequence, and has aldolase activity;(e) a DNA comprising the nucleotide sequence of base numbers 398 to 1141in the nucleotide sequence of SEQ ID No: 15;(f) a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of basenumbers 398 to 1141 in the nucleotide sequence of SEQ ID No: 15, andencodes a protein having aldolase activity;(g) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 16; and,(h) a DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 16, and has aldolase activity. Here, “one or several” refers to therange over which the steric structure of a protein of amino acidresidues or aldolase activity is not significantly impaired, and morespecifically, a range of 1 to 50, preferably 1 to 30 and more preferably1 to 10. Furthermore, “aldolase activity” refers to the activity thatsynthesizes IHOG from indole pyruvic acid and pyruvic acid (oroxaloacetic acid) as described above. However, in the case of an aminoacid sequence that contains a substitution, deletion, insertion,addition or inversion of one or several amino acid residues in the aminoacid sequence of SEQ ID No: 2, 3, or 16, it preferably retains aldolaseactivity of 10% or more, preferably 30% or more, more preferably 50% ormore, and still more preferably 70% or more, of protein having the aminoacid sequence of SEQ ID No: 2, 3, or 16 under conditions of 33° C. andpH 9.(2) Properties of Aldolase

Next, an explanation is provided of the properties of purified aldolasederived from Pseudomonas taetrolens strain ATCC4683 (PtALD) and purifiedaldolase derived from Pseudomonas coronafaciens strain AJ2791 (PcALD).

The PtALD of the present invention has the amino acid sequence of SEQ IDNo: 2 or 3 as is clearly determined by the previously described geneisolation and analysis. However, the present invention includes aprotein having an amino acid sequence that contains a substitution,deletion, insertion, addition or inversion of one or several amino acidresidues in the amino acid sequence of SEQ ID No: 2 or 3, which also hasaldolase activity.

The PcALD of the present invention has the amino acid sequence of SEQ IDNo: 16 as is clearly determined by the previously described geneisolation and analysis. However, the present invention includes aprotein having an amino acid sequence that contains a substitution,deletion, insertion, addition or inversion of one or several amino acidresidues in the amino acid sequence of SEQ ID No: 16, which also hasaldolase activity.

Namely, the aldolase of the present invention consists of the proteinsindicated in (i) to (l) below.

(i) a protein comprising the amino acid sequence of SEQ ID No: 2 or anamino acid sequence of residue numbers 5 to 225 of the same sequence;

(j) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one or aseveral amino acid residues in the amino acid sequence of SEQ ID No: 2or an amino acid sequence of residue numbers 5 to 225 in the samesequence, and has aldolase activity;(k) a protein comprising the amino acid sequence of SEQ ID No: 16; and,(l) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID No: 16,and has aldolase activity.

Here, the definitions of “several” and “aldolase activity” are the sameas defined in section (1), DNA Encoding Aldolase.

The aldolase of the present invention catalyzes the reaction thatsynthesizes 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG) byaldol condensation from indole pyruvic acid and pyruvic acid (oroxaloacetic acid).

The aldolase activity of the aldolase of the present invention may bemeasured by measuring the amount of IHOG formed from indole pyruvic acidand pyruvic acid (or oxaloacetic acid) by high-performance liquidchromatography (HPLC).

More specifically, aldolase activity may be estimated according to thefollowing steps of adding aldolase to a reaction solution composed of100 mM buffer, 50 mM indole-3-pyruvic acid, 250 mM pyruvic acid, 1 mMMgCl₂ and 1% (v/v) toluene; and shaking while reaction at 33° C. for 4hours; and then quantifying the amount of IHOG formed by HPLC.

HOG may be quantified by HPLC analysis using the “Inertsil ODS-2” (GLSciences Inc., 5 μm, 4.6×250 mm). The following indicates an example ofthe analysis conditions.

Mobile phase: 40% (v/v) acetonitrile/5 mM dihydrogen phosphatetetrabutyl ammonium solution

Flow rate: 1 ml/min

Column temperature: 40° C.

Detection: UV 210 nm

The aldolase of the present invention is able to catalyze the reactionthat synthesizes HOG by aldol condensation from indole pyruvic acid andpyruvic acid (or oxaloacetic acid). Although two examples of microbialenzymes capable of catalyzing aldol condensation using 2 molecules ofα-keto acid (or substituted α-keto acid) as a substrate have beenreported thus far consisting of 4-hydroxy-4-methyl-2-oxoglutaratealdolase derived from Pseudomonas species bacteria and4-hydroxy-2-oxoglutarate aldolase present in microorganisms such as E.coli and B. subtilis, there have been no findings or reports describingthe former acting on PHOG or HOG, and it is completely unknown as towhether it is possible to synthesize PHOG (and IHOG) by using thisenzyme. Furthermore, PHOG cleaving activity has not been observed forthe latter, and it has not been possible to synthesize PHOG (and IHOG)using this enzyme as well. Namely, the aldolase of the present inventiondiffers from aldolases that have been reported thus far in that it hasthe characteristic of being able to catalyze the reaction in which IHOGis synthesized by aldol condensation of indole pyruvic acid and pyruvicacid (or oxaloacetic acid).

Next, the following provides a description of the enzymatic propertiesinvestigated for purified PtALD.

PtALD catalyzes the reaction that forms a substituted α-keto acidrepresented by the following general formula (3′):

(wherein R¹ represents a hydrogen atom, an alkyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms, acarboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof; R³ represents a hydrogen atom or a carboxyl group,and R¹ may be substituted with at least one substituent selected fromthe group consisting of a halogen atom, a hydroxyl group, an alkyl grouphaving up to 3 carbon atoms, an alkoxy group having up to 3 carbon atomsand an amino group; and R⁴ represents a hydrogen atom, an alkyl grouphaving 1 to 8 carbon atoms, an alkoxyl group having 1 to 8 carbon atoms,a carboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof, and R⁴ may be substituted with at least onesubstituent selected from the group consisting of a halogen atom, ahydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxygroup having up to 3 carbon atoms and an amino group) from the compoundrepresented by the following general formula (1′):

(wherein R¹ and R³ has the same meaning as defined in general formula(3′)), and the substituted α-keto acid represented by the followinggeneral formula (2′):

(wherein R⁴ has the same meaning as defined in general formula (3′)).Thus, a process whereby the compound of general formula (3′) is producedfrom the compounds of general formula (1′) and general formula (2′) byusing the PtALD of the present invention also belongs to the presentinvention. Here, R⁴ is preferably a hydrogen atom or a carboxyl group.

The optimum pH of PtALD is nearly about 9 at 33° C. Furthermore, thePtALD of the present invention has pH stability at pH 6 or higher, andhas particularly high pH stability within the range of pH 6 to pH 11.Furthermore, the PtALD of the present invention has temperaturestability at 70° C. or lower, and has particularly high temperaturestability within the range of 20 to 60° C. Furthermore, the PtALD of thepresent invention has the property of aldolase activity being improvedby addition of inorganic phosphoric acid such as KPi to the enzymereaction mixture.

Since the molecular weight of PtALD is about 146 kDa as measured by gelfiltration and about 25 kDa as measured by SDS-PAGE, It was suggestedthat PtALD has a hexameric structure consisting of six subunits having amolecular weight of about 25 kDa.

(3) Process for Producing Aldolase

Next, an explanation is provided for the process of producing thealdolase of the present invention. There are two ways to produce thealdolase of the present invention. These consist of (i) a process ofcultivating an aldolase-producing microorganism to form and accumulatealdolase, and (ii) a process of preparing a transformant to formaldolase by a recombinant DNA technology and cultivating thetransformant to accumulate aldolase.

(i) Process for Forming and Accumulating Aldolase by MicrobialCultivation

Examples of microorganisms serving as acquisition sources of aldolase ina process for forming and accumulating aldolase by cultivatingaldolase-producing microorganisms include microorganisms belonging tothe genus Pseudomonas, Erwinia, Flavobacterium, and Xanthomonas.

Any microorganisms belonging to the genus Pseudomonas, Erwinia,Flavobacterium, or Xanthomonas may be used in the present inventionprovided they are microorganisms that form aldolase which catalyzes areaction that synthesizes a precursor keto acid (IHOG) from indolepyruvic acid and pyruvic acid (or oxaloacetic acid), and preferablemicroorganisms include Pseudomonas taetrolens ATCC4683, Pseudomonascoronafaciens AJ2791, Pseudomonas desmolytica AJ1582, Erwinia sp.AJ2917, Xanthomonas citri AJ2797 and Flavobacterium rhenanum AJ2468 isused preferably. Among these, Pseudomonas taetrolens ATCC4683 andPseudomonas coronafaciens AJ2791 are particularly preferable. Thelocations where these microorganisms are deposited are indicated below.

Pseudomonas coronafaciens strain AJ2791

(i) Deposit no.: FERM BP-8246 (transferred from FERM P-18881)

(ii) Deposition date: Jun. 10, 2002

(iii) Deposited location: National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depository (ChuoNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)

(2) Pseudomonas desmolytica strain AJ1582

(i) Deposit no.: FERM BP-8247 (transferred from FERM P-18882)

(ii) Deposition date: Jun. 10, 2002

(iii) Deposited location: National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depository (ChuoNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)

(3) Erwinia sp. strain AJ2917

(i) Deposit no.: FERM BP-8245 (transferred from FERM P-18880)

(ii) Deposition date: Jun. 10, 2002

(iii) Deposited location: National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depository (ChuoNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)

(4) Flavobacterium rhenanum strain AJ2468

(i) Deposit no.: FERM BP-1862

(ii) Deposition date: Sep. 30, 1985

(iii) Deposited location: National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depository (ChuoNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)

(5) Xanthomonas citri strain AJ2797

(i) Deposit no.: FERM BP-8250 (transferred from FERM P-8462)

(ii) Deposition date: Sep. 30, 1985

(iii) Deposited location: National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depository (ChuoNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)

Although the microorganism serving as the acquisition source of aldolasemay be cultivated in any form such as liquid cultivation and solidcultivation, an industrially advantageous method is deep-aerated stircultivation. Carbon sources, nitrogen sources, inorganic salts, andother trace nutrient elements commonly used in microbial cultivating maybe used as nutrient elements of nutritive media. All nutrient sourcesmay be used provided they may be used by the microbial strain beingused.

Aerobic conditions are used for the aeration conditions. The cultivatingtemperature may be sufficient within a range in which the microorganismsgrow and aldolase is produced. Thus, although the conditions are notstrict, the cultivating temperature is normally 10 to 50° C. andpreferably 30 to 40° C. The cultivating time varies according to othercultivating conditions. For example, the microorganisms may becultivated until the greatest amount of aldolase is produced, and thisis normally about 5 hours to 7 days, and preferably about 10 hours to 3days.

Following cultivation, the microbial cells are recovered bycentrifugation (e.g., 10,000×g for 10 minutes). Since the majority ofthe aldolase is present in the cells, the aldolase is solubilized bydisrupting or lysing the microbial cells. Ultrasonic disrupting, Frenchpress disrupting or glass bead disrupting may be used to disrupt themicrobial cells. In the case of lysing the cells, a method that uses eggwhite lysozyme, peptidase treatment, or a suitable combination thereofis adopted.

When aldolase derived from an aldolase-producing microorganism ispurified, although the aldolase is purified by using an enzymesolubilizing solution for the starting material, if undisrupted orunlysed residue remains, re-centrifuging the solubilization solution andremoving any residue that precipitates is advantageous to purification.

All commonly used methods for purifying ordinary enzymes may be employedto purify the aldolase, examples of which include ammonium sulfatesalting-out, gel filtration chromatography, ion exchange chromatography,hydrophobic chromatography and hydroxyapatite chromatography. As aresult, an aldolase-containing fraction with higher specific activitymay be obtained having higher specific activity.

(ii) Production Process Using Recombinant DNA Technology

Next, an explanation is provided for a process for producing aldolaseusing a recombinant DNA technology. There are numerous known examples ofproducing useful proteins such as enzymes and physiologically activesubstances using a recombinant DNA technology, and the use ofrecombinant DNA technology enables mass production of useful proteinspresent only in trace amounts in nature.

FIG. 1 is a flowchart of a process for producing the aldolase of thepresent invention.

First, a DNA is prepared that encodes the aldolase of the presentinvention (Step S1).

Next, the prepared DNA is ligated with a vector DNA to produce arecombinant DNA (Step S2), and cells are transformed by the recombinantDNA to produce a transformant (Step S3). Continuing, the transformant iscultivated in a medium, and the aldolase is allowed to form andaccumulate in any one of the medium and cells or both (Step S4).

Subsequently, the process proceeds to Step S5 where purified aldolase isproduced by recovering and purifying the enzyme.

The desired substituted α-keto acid may be produced in a large amount byusing the purified aldolase produced at Step S5 or any of the medium andcells or both in which aldolase has accumulated at Step S4 in an aldolreaction (Step S6).

The DNA that is ligated with the vector DNA may allow expression of thealdolase of the present invention.

Here, examples of aldolase genes ligated into the vector DNA include thepreviously described DNA indicated below:

(a) a DNA comprising the nucleotide sequence of SEQ ID No: 1, or thenucleotide sequence of base numbers 444 to 1118 or base numbers 456 to1118 in the same sequence;

(b) a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 1 or a nucleotide sequence of base numbers 444 to 1118 or 456 to1118 of the same sequence, and encodes a protein having aldolaseactivity;(c) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 2 or an amino acid sequence of residue numbers 5 to 225 ofthe same sequence;(d) a DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 2 or an amino acid sequence of residue numbers 5 to 225 of the samesequence, and has aldolase activity;(e) a DNA comprising the nucleotide sequence of SEQ ID No: 15, or anucleotide sequence of base numbers 398 to 1141 of the same sequence;(f) a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 15 or a nucleotide sequence of base numbers 398 to 1141 of the samesequence, and encodes a protein having aldolase activity;(g) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No: 16; and,(h) a DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition, or inversion ofone or several amino acid residues in the amino acid sequence of SEQ IDNo: 16, and has aldolase activity.

In the case of mass production of protein using recombinant DNAtechnology, it is preferable to form a inclusion body of protein byassociating the protein within a transformant that produces the protein.The advantages of this expression production method include being ableto protect the desired protein from digestion by proteases presentwithin microbial cells, and being able to easily purify the desiredprotein by a centrifugation procedure after disrupting the microbialcells.

Protein inclusion bodies that have been obtained in this manner aresolubilized by a protein denaturant, and after going through arenaturing procedure consisting primarily of removing the denaturant,such protein is converted to properly folded, physiologically activeprotein. There are numerous examples of this such as the restoration ofthe activity of human interleukin-2 (JP-A-S61-257931).

In order to obtain a active protein from a inclusion body of protein, aseries of procedures including solubilization and restoration ofactivity are required, and these procedures are more complex thanproducing the active protein directly. However, in the case of largescale production in microbial cells of a protein that affects microbialgrowth, those effects may be inhibited by allowing the protein toaccumulate within the microbial cells in the form of inactive inclusionbodies.

Methods for mass production of a desired protein in the form ofinclusion bodies include a method in which the desired protein isexpressed independently under the control of a powerful promoter, and amethod in which the desired protein is expressed as a fused protein witha protein that is known to be expressed in large scale.

Furthermore, it is also effective to arrange the recognition sequence ofa restricting protease at a suitable location in order to cut out thedesired protein after having expressed in the form of a fused protein.

In the case of large scale protein production using recombinant DNAtechnology, cells such as bacterial cells, Actinomyces cells, yeastcells, mold cells, plant cells and animal cells may be used for the hostcells that are transformed. Examples of bacterial cells for whichhost-vector systems have been developed include Escherichia species,Pseudomonas species, Corynebacterium species, and Bacillus species, andpreferably Escherichia coli is used. This is because there are numerousknowledge regarding technologies for mass production of protein usingEscherichia coli. The following provides an explanation of a process forproducing aldolase using transformed E. coli.

A promoter normally used for heterogeneous protein production in E. colimay be used for the promoter that expresses a DNA encoding aldolase,examples of which include powerful promoters such as T7 promoter, trppromoter, lac promoter, tac promoter and PL promoter.

In order to produce aldolase in the form of a fused protein inclusionbody, a gene that encodes another protein, preferably a hydrophilicpeptide, is ligated either upstream or downstream of the aldolase genewith a fused protein gene. The gene that encodes another protein may bea gene that increases the amount of fused protein accumulated andenhances the solubility of the fused protein following the denaturationand regeneration steps, examples of candidates for which include 17 gene10, β-galactosidase gene, dehydrofolate reductase gene, interferon-γgene, interleukin-2 gene and prochymosin gene.

When ligating these genes with a gene that encodes aldolase, the codonreading frames are made to match. The genes may either be ligated in asuitable restriction enzyme site or using a synthetic DNA of anappropriate sequence.

In order to increase the amount produced, it is preferable to couple atranscription terminating sequence in the form of a terminatordownstream from the fused protein gene. Examples of this terminatorinclude T7 terminator, fd phage terminator, T4 terminator, tetracyclineresistance gene terminator, and E. coli trpA gene terminator.

Multi-copy vectors are preferable for the vector used to introduce agene that encodes aldolase or a fused protein of aldolase with anotherprotein into E. coli, examples of which include plasmids having areplication starting point derived from Col E1 such as pUC plasmids,pBR322 plasmids or their derivatives. A “derivative” here refers to thathas undergone alteration of a plasmid by base substitution, deletion,insertion, addition or inversion. The alteration referred to hereincludes alteration caused by mutagenic treatment using a mutagen or UVirradiation or by spontaneous mutation.

It is preferable that the vector has a marker such as ampicillinresistance gene in order to select the transformant. Examples of suchplasmids include commercially available expression vectors having apowerful promoter (such as pUC (Takara), pPROK (Clontech), and pKK233-2(Clontech)).

A recombinant DNA is obtainable from ligating a DNA fragment, in which apromoter, a gene encoding aldolase or fused protein consisting ofaldolase and another protein, and a terminator are ligated in thatorder, with a vector DNA.

When E. coli is transformed using the recombinant DNA and that E. coliis then cultivated, aldolase or a fused protein of aldolase with anotherprotein is expressed and produced. A strain that is normally used forexpression of heterogeneous genes may be used for the transformed host,and E. coli strain JM109(DE3) and E. coli strain JM109 are particularlypreferable. The transformation method and method for selecting thetransformant are described in, for example, Molecular Cloning, 2ndedition, Cold Spring Harbor Press (1989).

In the case of expressing as fused protein, the aldolase may be cut outusing a restricting protease such as blood coagulation factor Xa orkallikrein that recognizes sequences not existing in aldolase as therecognition sequence.

A medium normally used for cultivating E. coli may be used for theproduction medium, examples of which include M9-casamino acid medium andLB medium. Furthermore, conditions of cultivating and productioninduction may be appropriately selected according to the type of themarker and promoter of the vector used, and type of host microorganismused.

The following method may be used to recover the aldolase or fusedprotein of aldolase with another protein. If the aldolase or its fusedprotein is solubilized within microbial cells, then it may be used inthe form of a crude enzyme solution after recovering the microbial cellsand disrupting or lysing the recovered cells. Furthermore, the aldolaseor its fused protein may also be used after purifying by precipitation,filtration, column chromatography or other common technique asnecessary. In this case, a purification method may also be used thatuses an antibody of the aldolase or its fused protein.

When a protein inclusion body is formed, it is solubilized with adenaturant. Although it may be solubilized together with microbial cellprotein, in consideration of the subsequent purification procedure, itis preferable to take out the inclusion body and then solubilize it. Amethod known in the prior art may be used to recover the inclusion bodyfrom the microbial cell. For example, the inclusion body may berecovered by disrupting the microbial cell followed by centrifugalseparation. Examples of denaturants that solubilize protein inclusionbodies include guanidine hydrochloride (e.g., 6 M, pH 5-8) and urea(e.g., 8 M).

The protein inclusion body may be regenerated as active protein byremoving these denaturants by treatment such as dialysis. Dialysissolutions such as Tris-HCl buffer or phosphate buffer may be used fordialysis, and the concentration may be from 20 mM to 0.5 M, and the pHmay be from pH 5 to pH 8.

The protein concentration during the regeneration step is preferablyheld to about 500 μg/ml or less. In order to prevent the regeneratedaldolase from undergoing self-crosslinking, the dialysis temperature ispreferably 5° C. or lower. Furthermore, activity may also be expected tobe restored by other methods used to remove denaturant such as dilutionand ultrafiltration in addition to the aforementioned dialysis.

When the aldolase gene is derived from bacteria belonging to the genusPseudomonas, the aldolase may be expressed and produced by using thePseudomonas species bacteria as a host in one preferable mode.Descriptions of examples of host cells in this case include a report ona recombinant expression method in Pseudomonas syringae by Shi-En Lu etal. (FEMS Microbiology Letters 210 (2002) p. 115-121), a report on arecombinant expression method in Pseudomonas aeruginosa by Olsen, R. H.et al. (Journal of Bacteriology, (1982) 150, p. 60-69) and a report on arecombinant expression method in Pseudomonas stutzeri by Grapner, S. etal. (Biomol. Eng., (2000), 17, p. 11-16). However, the Pseudomonasspecies bacteria used as host cells for expressing aldolase are notlimited to those recited herein.

Next, with respect to the vector used to introduce aldolase gene intoPseudomonas species bacteria, a plasmid may be used that has areplication starting point which function inside Pseudomonas speciesbacterial cells. For example, plasmid pKLH4.05 has been reported by EzaKalyaeva et al. to have a replicon TFK that function in Pseudomonasaeruginosa. Wide-spectrum host vectors may also be used that are used totransform Gram-negative bacteria. These vectors are known to function inPseudomonas species bacteria as well, examples of which include RK404(Dicta, G et al., Plasmid 13 (1985) p. 149-153) and RSF1010 (Frey, J. etal., Gene 24 (1982) p. 289-296).

In the case of using the DNA indicated in SEQ ID No: 1 for the DNA thatencodes aldolase, aldolase is produced that has the amino acid sequenceof SEQ ID No: 2 or 3, and in the case of using the DNA indicated in SEQID No: 15, aldolase is produced that has the amino acid sequence of SEQID No: 16.

[II] Method for Producing Substituted α-Keto Acid Using Aldolase

Next, a description is provided of the method for producing substitutedα-keto acid of the present invention. The method for producingsubstituted α-keto acid of the present invention is a method forproducing the substituted α-keto acid represented by the followinggeneral formula (3):

by reacting the substituted α-keto acid represented by the followinggeneral formula (1):

with the substituted α-keto acid represented by the following generalformula (2):

wherein the reaction is performed in the presence of a protein thatcatalyzes the reaction.

In general formula (1), R¹ represents an alkyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms, acarboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof. R¹ may also be substituted with a halogen atom, ahydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxygroup having up to 3 carbon atoms or an amino group. R¹ is preferably asubstituent having 4 or more carbon atoms and preferably 6 or morecarbon atoms, while an aryl group or an aralkyl group having up to 20carbon atoms (aryl groups such as a phenyl group, a tolyl group, a xylylgroup, a biphenyl group, a naphthyl group, an anthryl group or aphenantolyl group; an aralkyl groups such as a benzyl group, abenzhydryl group, a styryl group, a phenethyl group, a trityl group or acinnamyl group), and a heterocycle-containing hydrocarbon group havingup to 11 carbon atoms (heterocyclic groups such as a furyl group, athienyl group, a pyridyl group, a piperidyl group, a piperidino group, amorpholino group or an indolyl group; alkyl groups substituted by theseheterocyclic groups) are particularly preferable. R¹ is particularlypreferably a benzyl group or a 3-indolylmethyl group, and a3-indolylmethyl group is the most preferable. Namely, phenyl pyruvicacid or indole pyruvic acid is preferable for the substituted α-ketoacid of general formula (1), and indole pyruvic acid is particularlypreferable.

In general formula (2), R² represents an alkyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms, acarboxyalkyl group having 2 to 9 carbon atoms, an aryl or an aralkylgroup having up to 20 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a hydroxyl group or an esterderivative thereof. When R² contains an aromatic ring or a hetero ring,the aromatic ring or hetero ring may be substituted with at least onetype of substituent selected from the group consisting of a halogenatom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, analkoxy group having up to 3 carbon atoms and an amino group. However,when R¹ represents a hydrogen atom, a methyl group or a carboxymethylgroup in general formula (1), R² does not represent a hydrogen atom. R²is preferably a hydrogen atom or a carboxyl group, and particularlypreferably a hydrogen atom. Namely, the substituted α-keto acid ofgeneral formula (2) is preferably oxaloacetic acid or pyruvic acid, andparticularly preferably pyruvic acid.

In general formula (3), R¹ and R² have the same meanings as R¹ and R² ingeneral formulas (1) and (2).

In the production process of a substituted α-keto acid of the presentinvention, it is most preferable to synthesize IHOG shown in thefollowing formula (4) by using indole pyruvic acid for the substitutedα-keto acid of general formula (1), and using pyruvic acid for thesubstituted α-keto acid of general formula (2).

There are no particular limitations on the protein that catalyzes thereaction, and any protein may be used provided it is a protein that iscapable of catalyzing a reaction that synthesizes the substituted α-ketoacid represented by the general formula (3) by aldol condensation of thesubstituted α-keto acid represented by the general formula (1) and thesubstituted α-keto acid represented by the general formula (2). Namely,as long as the protein catalyzes the reaction, it may be a proteinderived from a microorganism or a protein synthesized by a chemicalsynthesis process.

A preferable example of such a protein is the aldolase explained insection [1] describing aldolase. In this case, aldolase may be usedwhich has been obtained by cultivating microbial cells that form proteinthat catalyzes the reaction (aldolase) among microorganisms belonging tothe genus Pseudomonas, Erwinia, Flavobacterium, or Xanthomonas, or (2)aldolase may be used which has been obtained by producing a transformantthat forms a protein that catalyzes the reaction using recombinant DNAtechnology followed by cultivating the transformant.

The protein that catalyzes the reaction may be added in any form to thereaction mixture provided it is able to catalyze the reaction thatsynthesizes the aforementioned substituted α-keto acid represented bygeneral formula (3). Namely, the protein that catalyzes the reaction maybe added to the reaction mixture by itself, or it may be added to thereaction mixture in the form of a composition having aldolase activitythat contains a protein that catalyzes the reaction (aldolase).

A “composition having aldolase activity” here is only required to be onewhich contains a protein that catalyzes the reaction (aldolase), andspecific examples include a culture, medium (in which microbial cellshave been removed from a culture), microbial cells (including bothcultivated microbial cells and washed microbial cells), a treatedmicrobial cell product that have been disrupted or lysed, and acomposition having aldolase activity obtainable from purifying any ofthe medium and cells or both (crude enzyme solution, purified enzyme).For example, in the case of producing a substituted α-keto acid usingaldolase-producing microorganisms or cells that have been transformed bya recombinant DNA, the substrate may be added directly to the culturewhile cultivating, or may be used in the form of microbial cells orwashed microbial cells that have been separated from the culture.Furthermore, a treated microbial cell product that have been disruptedor lysed may be used directly, or the aldolase may be recovered from thetreated microbial cell product and used as a crude enzyme solution, orused after purifying the enzyme. Namely, as long as it is in the form ofa fraction that has aldolase activity, it may be used in the process forproducing a substituted α-keto acid of the present invention in anyform.

In order to perform an aldol reaction using aldolase or a compositionhaving aldolase activity, a reaction solution containing a substitutedα-keto acid represented by the general formula (1), a substituted α-ketoacid represented by the general formula (2) and a protein oraldolase-containing composition that catalyzes the reaction is adjustedto a suitable temperature of 20 to 50° C. and allowing to standundisturbed, shaking or stirring for 30 minutes to 5 days whilemaintaining at pH 6 to 12.

The reaction velocity may also be improved by adding a bivalent cationsuch as Mg²⁺, Mn²⁺, Ni²⁺, or Co²⁺ to the reaction mixture. Mg²⁺ may beused preferably in terms of cost and so forth.

When adding these bivalent cations to the reaction solution, althoughany salt may be used provided it does not hinder the reaction, MgCl₂,MgSO₄, MnSO₄, and so forth may be used preferably. The concentrations ofthese bivalent cations may be determined by simple preliminary studiesconducted by a person with ordinary skill in the art. These bivalentcations may be added within the range of 0.01 mM to 10 mM, preferably0.1 mM to 5 mM, and more preferably 0.1 mM to 1 mM.

As a preferable example of reaction conditions when performing theprocess for producing substituted α-keto acid of the present invention,an enzyme source in the form of washed cells of aldolase-expressing E.coli are added at 10% (w/v) to a reaction solution consisting of 100 mMbuffer, 50 mM indole-3-pyruvic acid, 250 mM pyruvic acid, 1 mM MgCl₂ and1% (v/v) toluene followed by reacting while shaking at 33° C. for 4hours to obtain 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG).

The substituted α-keto acid of general formula (3) that is formed may beseparated and purified according to known techniques. Examples of themethod may include a method in which the substituted α-keto acid iscontacted with an ion exchange resin to adsorb basic amino acidsfollowed by elution and crystallization, and a method in which theproduct obtained by elution is discolored and filtrated with activatedcharcoal followed by crystallization to obtain substituted α-keto acid.

Use of the process for producing substituted α-keto acid of the presentinvention makes it possible to form a precursor keto acid (IHOG) ofmonatin from indole pyruvic acid and oxaloacetic acid (or pyruvic acid).Since IHOG may be used to derive monatin by aminating position 2, it isuseful as an intermediate in monatin synthesis.

EXAMPLES

The present invention will be explained in further detail with referenceto examples shown below, however, the invention is not limited thereto.In the examples, IHOG and PHOG used as substrates were synthesizedaccording to the processes described in Reference Examples 1 and 2.

Example 1 [I] Screening of Microorganisms Having Aldolase Activity forPHOG

Microbial strains having aldolase activity using4-phenylmethyl-4-hydroxy-2-oxoglutarate (PHOG) as a substrate werescreened for.

Microorganisms (bacteria, yeast) were inoculated onto bouillon platemedium (Eiken Chemical Co., Ltd.) and cultivated at 30° C. for 24 hours.The microorganisms were then inoculated onto plates containing 0.5 g/dlglycerol, 0.5 g/dl fumaric acid, 0.3 g/dl yeast extract, 0.2 g/dlpeptone, 0.3 g/dl ammonium sulfate, 0.3 g/dl K₂HPO₄, 0.1 g/dl KH₂PO₄,0.05 g/dl MgSO₄.7H₂O, 0.25 g/dl sodium phthalate and 2 g/dl agar powder(pH 6.5) followed by cultivating at 30° C. for 24 hours. The resultingmicrobial cells were inoculated into a reaction solution comprised of100 mM Tris-HCl (pH 8.0), 50 mM PHOG, 1 mM MgCl₂, 5 mM potassiumphosphate solution (KPi) and 1% (v/v) toluene to wet cells weight ofabout 1% (w/v), and incuvated at 30° C. for 24 hours. The concentrationof free pyruvic acid in the reaction solution was quantified by anenzymatic method using lactate dehydrogenase (LDH). 10 μl of sample wasthen added to 200 μl of a reaction solution comprised of 100 mM Tris-HCl(pH 8.0), 1.5 mM NADH, 5 mM MgCl₂ and 25 U/ml LDH followed by incubatingat 30° C. for 10 minutes. The optical absorbance at 340 nm was measuredafter the reaction, and the amount of pyruvic acid in the sample wasquantified from the reduction in the amount of NADH.

The amount of phenyl pyruvic acid formed was quantified by HPLC analysisusing an “Inertsil ODS-2” column (GL Sciences Inc., 5 μm, 4.6×250 mm).The analysis conditions were as indicated below.

Mobile phase: 20% (v/v) acetonitrile/0.05% (v/v) aqueous trifluoroaceticacid

Flow rate: 1 mL/min

Column temperature: 40° C.

Detection: UV 210 nm

Under these conditions, PHOG was eluted at retention time of about 9.8minutes while phenyl pyruvic acid was eluted at a retention time ofabout 12 minutes, and each was able to be separated and quantified.

The value obtained by subtracting the amount of pyruvic acid or phenylpyruvic acid formed from PHOG in a reference (microorganism non-additionreaction) from the amount formed in a test microorganism additionreaction was used to indicate the amount formed by aldolase. As aresult, aldolase activity using PHOG as substrate was found for themicrobial strains listed in Table 1.

TABLE 1 Results of Screening for Microbial Strains Having AldolaseActivity for PHOG Pyruvic Phenylpyruvic acid acid Microbial strain (mM)(mM) Pseudomonas taetrolens ATCC4683 34.9 35.0 Pseudomonas coronafaciensAJ2791 33.6 33.9 Pseudomonas desmolytica AJ1582 1.1 2.9 Erwinia sp.AJ2917 0.8 3.0 Flavobacterium rhenanum CCM298 AJ2468 3.0 6.1 Xanthomonascitri AJ2797 1.0 3.2

PHOG was synthesized from phenyl pyruvic acid and oxaloacetic acid orpyruvic acid using Pseudomonas taetrolens ATCC4683 cells. Microbialcells of P. taetrolens ATCC4683 (AJ2212) were inoculated to a finalconcentration of about 1% (w/v) into a reaction solution comprised of100 mM Tris-HCl (pH 8.0), 50 mM phenyl pyruvic acid, 1 mM MgCl₂, 5 mMKPi, 100 mM oxaloacetic acid, or pyruvic acid and 1% (w/w) toluene,followed by incuvation at 30° C. for 16 hours. After the reaction, theamount of PHOG formed was quantified by HPLC. The amounts of PHOG formedfrom phenyl pyruvic acid and oxaloacetic acid or pyruvic acid are shownin Table 2.

TABLE 2 Amounts of PHOG Formed from Phenyl Pyruvic Acid and OxaloaceticAcid and/or Pyruvic Acid Oxaloacetic acid lot Pyruvic acid lotMicroorganism addition lot 14.3 (mM) 9.3 (mM) Control (Mg addition) lot8.6 1.7 Control (Mg non-addition) lot trace N.D.

According to Table 2, an increase in the amount of PHOG formed wasobserved in the microorganism addition reaction, and PHOG was determinedto be able to be formed by the action of aldolase for both thecombinations of phenyl pyruvic acid+oxaloacetic acid and phenyl pyruvicacid+pyruvic acid.

[II] Purification of IHOG-Aldolase Derived from Pseudomonas taetrolensstrain ATCC4683

IHOG-aldolase was purified as described below from a soluble fraction ofP. taetrolens strain ATCC4683. Aldolase activity using PHOG as asubstrate was measured under the following conditions.

Reaction conditions: 50 mM Tris-HCl (pH 8.0), 2 mM PHOG, 0.2 mM NADH,0.2 mM KPi, 1 mM MgCl₂, 16 U/ml of lactate dehydrogenase and 3 μLenzyme/600 μl reaction solution, optical absorbance at 340 nm measuredat 30° C.

(1) Preparation of Soluble Fraction

A loopful of P. taetrolens ATCC4683 cells cultivated at 30° C. for 24hours on bouillon plate medium was picked from the plate, and inoculatedinto a 500 ml volumetric flask containing 50 ml of enzyme productionmedium (0.5 g/dl glycerol, 0.5 g/dl fumaric acid, 0.5 g/dl ammoniumsulfate, 0.3 g/dl K₂HPO₄, 0.1 g/dl KH₂PO₄, 0.05 g/dl MgSO₄.7H₂O, 0.3g/dl yeast extract, 0.2 g/dl peptone, 0.25 g/dl sodium phthalate and0.005% Antifoam A (Sigma), adjusted to pH 6.5 with KOH) followed byshaken-culturing at 30° C. for 24 hours. 0.5 ml of the culture wasinoculated into 500 ml volumetric flasks containing 50 ml of enzymeproduction medium followed by shake culture at 30° C. for 24 hours. Themicroorganisms were collected from the resulting culture (total 2 L) bycentrifugation, and after washing by suspending in buffer A (20 mMTris-HCl (pH 7.6)), were again recovered by centrifugation. Theresulting washed microbial cells were suspended in 200 ml of buffer Aand then ultrasonically disrupted at 4° C. for 30 minutes. The microbialcell residue was removed from the disruption solution by centrifugation(×8000 rpm, 10 min×2 times) followed by additional ultracentrifugation(×50000 rpm, 30 min). After the ultracentrifugation, the resultingsupernatant was obtained as the soluble fraction.

(2) Anionic Exchange Chromatography: Q-Sepharose FF

80 ml of the aforementioned soluble fraction was applied to an anionicexchange chromatography column, Q-Sepharose FF 26/10 (Pharmacia, CV=20ml) equilibrated with buffer A, and adsorbed onto the carrier. Afterwashing out any unbound proteins using buffer A, the adsorbed proteinwas eluted using liner gradient of the KCl concentration from 0 M to 0.7M (total: 140 ml). aldolase activity for PHOG was detected in thefraction equivalent to about 0.5 M. The same chromatography procedurewas repeated twice.

(3) Hydrophobic Chromatography: Phenyl Sepharose HP HR 16/10

The fraction in which aldolase activity had been detected was dialyzedat 4° C. overnight against buffer B (50 mM Tris-HCl (pH 7.6), 1 Mammonium sulfate, pH 7.6) and then filtered with a 0.45 μm filter. Theresulting filtrate was applied to a hydrophobic chromatography column,Phenyl Sepharose HP HR 16/10 (Pharmacia) equilibrated with buffer B. Thealdolase was adsorbed to the carrier.

After washing out any unbound proteins using buffer B, aldolase waseluted using liner gradient of the ammonium sulfate concentration from 1M to 0 M. The aldolase activity was detected in the elution fractionwhere the concentration of ammonium sulfate was about 0.2 M.

(4) Gel Filtration Chromatography: Sephadex 200 HP 16/60

Each fraction containing aldolase was collected, dialyzed against bufferA and filtered with a 0.45 μm filter. The resulting filtrate wasconcentrated using the Centriprep 10 ultrafiltration membrane. Theresulting concentrate was applied to a gel filtration chromatographycolumn, Sephadex 200 HP 16/60 (Pharmacia) equilibrated with buffer C (20mM Tris-HCl (pH 7.6), 0.1 M KCl), and eluted at a flow rate of 1 mL/min.the aldolase was eluted in the fraction from 66 to 71 ml. The molecularweight of the aldolase as determined from the elution position of peakactivity was estimated to be about 146 kDa.

(5) Anionic Exchange Chromatography: Mono Q HR5/5

The resulting fraction was filtered with a 0.45 μm filter. Here, theresulting filtrate was applied to an anionic exchange chromatographycolumn, Mono Q HR 5/5 (Pharmacia) equilibrated with buffer A. Thealdolase was adsorbed to the carrier. After washing out any unboundproteins with buffer A, protein was eluted using liner gradient of theKCl concentration from 0 mM to 700 mM (total: 24 ml). The aldolaseactivity was measured for each eluted fraction, and aldolase activitywas observed at the elution position where the KCl concentration wasabout 0.4 M.

(6) Hydroxyapatite Chromatography: CHT-II

The resulting fraction was dialyzed at 4° C. overnight against buffer D(10 mM potassium phosphate buffer (pH 7.0)), and filtered with a 0.45 μmfilter. Here, the resulting filtrate was applied to a hydroxyapatitechromatography column, 5 ml CHT-II (BioRad) equilibrated with buffer D.The aldolase activity was detected at the unbound fraction, aldolase wasable to be separated from the adsorbed protein.

When the fraction purified according to the column chromatographyprocedures described above was applied to SDS-PAGE, a nearly single bandwas detected at the location corresponding to about 25 kDa. Since theestimated molecular weight as determined by gel filtrationchromatography was about 146 kDa, it was suggested that the aldolaseforms a hexamer. The purification table is shown in Table 3.

TABLE 3 Purification Table of IHOG Aldolase Derived from Pseudomonastaetrolens Strain ATCC4683 specific purifi- total protein activitycation activity yield (mg) (U/mg) fold (U) (%) soluble fraction 37500.014 1 51 100 Q-sepharose HP 26/10 510 0.060 4.4 30.5 59.8 Phenylsepharose HP 16/10 21.2 0.893 66 19.0 37.2 Sephadex200 HP 16/60 1.94.643 341 8.65 17.0 monoQ HR5/5 0.49 10.89 800 5.33 10.4 HydroxyapatiteCHT-II 0.025 28.70 2110 0.71 1.4

[III] Determination of Internal Amino Add Sequence of IHOG Aldolase

After applying an approximately 2 μg aliquot of the purified aldolase toSDS-PAGE, the sample in the SDS-PAGE gel was treated with trypsin (pH8.5, 35° C., 20 hours) and then applied to reverse phase HPLC toseparate the fragment peptides. Amino acid sequences consisting of 20residues and 12 residues, respectively (SEQ ID Nos: 4 and 5), weredetermined as shown below for two of the separated fractions.

TABLE 4 Determined Internal Amino Acid Sequences SEQ ID No. 4SLLDA FQNVV TPHIS DNLGR SEQ ID No. 5 AEIAT GALDQ SW

[IV] Cloning of IHOG-Aldolase Gene Derived from P. taetrolens StrainATCC4683

(1) Preparation of Chromosomal DNA

P. taetrolens strain ATCC4683 was cultivated at 30° C. overnight with 50ml of bouillon medium (pre-cultivation). 5 ml of this culture wasinoculated into the 50 ml of fresh bouillon medium. After thecultivation until the late logarithmic growth phase, 50 ml of culturewas centrifuged (12000×g, 4° C., 15 minutes) to recover themicroorganisms. A chromosomal DNA was then prepared according toestablished methods using these microbial cells.

(2) Cloning of a Internal DNA of the Aldolase Gene by PCR

The following mixed primers (SEQ ID Nos: 6 and 7) were synthesized basedon the internal amino acid sequence determined for IHOG-aldolase.

TABLE 5 Mixprimers Designed and Synthesized Basedon Internal Amino Acid Sequence SEQ ID No. 6TTY CAR AAY GTS GTS ACS CCS C SEQ ID No. 7TGR TCR ATN GCN CCS GTN GCR ATY TCN GC

Amplification was performed by PCR with the synthesized mixed primersand the chromosomal DNA of P. taetrolens ATCC4683 as a template. The PCRreaction was performed with the PCR Thermal PERSONEL (Takara) for 30cycles under the conditions indicated below.

94° C., 30 seconds

55° C., 30 seconds

72° C., 1 minute

The PCR product was applied to agarose gel electrophoresis, and anapproximately 500 bp fragment was amplified. This DNA fragment wascloned to pUC18, followed by determination of the nucleotide sequence.The amino acid sequence estimated from the obtained DNA fragment matchedwith the internal amino acid sequence of IHOG aldolase, therebyconfirming that the desired aldolase gene had been obtained.

(3) Cloning of the Full Length Gene by Colony Hybridization

The full-length gene was cloned by Southern hybridization analysis andcolony hybridization with the DNA fragment amplified by PCR. The DNAprobe was produced by using DIG High Prime (Roche Diagnostics K.K.),incubating overnight (i.e., O/N) at 37° C. according to the instructionmanual to label the probe. Southern hybridization analysis was performedaccording to the manual by digesting 1 μg of chromosomal DNA with eachof restriction enzyme, electrophoresing in 0.8% agarose gel and blottingonto a nylon membrane. Hybridization was performed using DIG Easy Hyb(Roche Diagnostics K.K.) by pre-hybridizing at 50° C. for 1 hour, addingthe probe and then hybridizing with O/N. Bands were detected using theDIG Nucleotide Detection Kit. As a result, an approximately 4 kbp Pstlfragment was detected that strongly hybridized by using the PCR fragmentas a probe. Next, this Pstl fragment was obtained by colonyhybridization. 20 μg of chromosomal DNA was treated with Pstl followedby applying to agarose gel electrophoresis and recovering a fragment ofwhich size was about 4 kbp. This was then ligated into pUC118 to producea library in E. coli JM109. The colonies were then transferred to anylon membrane fitter (Hybond-N, Amersham) followed by alkalinedenaturation, neutralization and immobilization treatment. Hybridizationwas performed using DIG Easy Hyb. The filter was immersed in a bufferand pre-hybridized at 42° C. for 1 hour. Subsequently, the previouslyprepared labeled probe was added, followed by hybridization at 42° C.for 16 hours. After washing with SSC, colonies that hybridized with theprobe were detected using the DIG Nucleotide Detection Kit (RocheDiagnostics K.K.). As a result, a clone was obtained that stronglyhybridized with the probe.

The nucleotide sequence of plasmid DNA recovered from the obtained clonewas determined, and it was clearly demonstrated to have the nucleotidesequence of SEQ ID No: 1. An orf of 678 bp that contained the nucleotidesequence corresponding to the determined amino acid sequence (numbers507 to 566 and numbers 1046 to 1082 in SEQ ID No: 1) was found and theentire length of the desired aldolase gene was obtained.

(4) Expression of IHOG-Aldolase in E. coli/(Part 1)

The fragment amplified using the primers shown in Table 6 (SEQ ID Nos: 8and 9) from a chromosomal DNA of P. taetrolens ATCC4683 was digestedwith BamHI/HindIII, inserted into the BamHI/HindIII site of pUC18, toconstruct plasmid pUCALD. E. coli JM109 was transformed with thusobtained pUCALD, and the transformant was cultured at 37° C. overnightin LB medium containing 50 μg/ml of ampicillin (pre-cultivation). Thepre-culture was inoculated at 1% in 50 ml of LB medium followed bycultivating at 37° C. IPTG was added to a final concentration of 1 mM atabout 2 hours after the start of cultivating, followed by additionallycultivating for 3 hours. After cultivating, the microorganisms wererecovered, washed, and then suspended in 1 ml of 20 mM Tris-HCl (pH 7.6)followed by disrupting the microbial cells using a Multi-Beads Shocker(Yasui Kikai Corporation). The crude extract was then centrifuged at15000 rpm for 10 minutes, and the resulting supernatant was used as acrude enzyme solution.

TABLE 6 Primer SEQ ID No. 8 ALD-5′ Bam(5′-GCC GGA TCC ACA AGGGTT CAG TCA TTC ATG G-3′) SED ID No. 9 ALD-3′Hind(5′-CCG AAG CTT TCA GTT CGC CAG GCC AGC C-3′)

When aldolase activity was measured using the crude enzyme solution andPHOG as a substrate, in contrast to aldolase activity for PHOG not beingdetected in the E. coli harboring pUC18 (control), aldolase activity forPHOG of 0.81 U/mg protein was detected in the strain harboring pUCADL.As a result, the gene was demonstrated to encode the desired aldolase.

[V] Synthesis of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG)from Indole-3-pyruvic Acid and Pyruvic Acid Using an Aldolase-ExpressingStrain

4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG) was synthesizedfrom indole-3-pyruvic acid and pyruvic acid with washed cells of thealdolase-expressing E. coli produced in [IV] as the enzyme source. Theamount of IHOG was determined by HPLC analysis using the “InertsilODS-2” (GL Sciences Inc., 5 μm, 4.6×250 mm). The analysis conditions areas indicated below.

Mobile phase: 40% (v/v) acetonitrile/5 mM tetrabutyl

ammonium dihydrogen phosphate solution

Flow rate: 1 ml/min

Column temperature: 40° C.

Detection: UV 210 nm

Washed cells of aldolase-expressing E. coli were added at 10% (w/v) to areaction solution comprised of 100 mM buffer (Tris-HCl 8.0, 9.0, orglycine-NaOH 10.0) 50 mM indole-3-pyruvic acid, 250 mM pyruvic acid, 1mM MgCl₂ and 1% (v/v) toluene, and allowed to react by shaking at 33° C.for 4 hours. The enzyme reaction solution was suitably diluted and theamount of HOG formed was quantified.

TABLE 7 Amount of IHOG Formed by Aldolase pH aldolase IHOG (mM) 8 + 9.28 − 0.42 9 + 12.1 9 − 1.6 10 + 10.7 10 − 5.4

As a result, the amount of IHOG produced increased in thealdolase-expressing E. coli addition group, and IHOG was formed by thealdolase.

Example 2 High-Level Expression of IHOG-Aldolase in E. Coli (Part 2)

(1) Construction of Plasmid pTrp4 Containing trp Promoter and rmBTerminator

The promoter region of the trp operon in a chromosomal DNA of E. coliW3310 by PCR with the oligonucleotides shown in Table 8 as primers(combination of SEQ ID No: 10 and 11), and the resulting DNA fragmentwas ligated into a pGEM-Teasy vector (Promega). E. coli JM109 was thentransformed with this ligation mixture, and a strain was selected amongampicillin-resistant strains that had the desired plasmid in which thetrp promoter was inserted in the opposite orientation of the lacpromoter. Next, the DNA fragment containing trp promoter obtained bytreating this plasmid with Eco01091/EcoRI was ligated with the productof treating pUC19 (Takara) with Eco01091/EcoRI. E. coli JM109 was thentransformed with this ligation mixture, a strain was selected amongampicillin-resistant strains that had the desired plasmid, and thatplasmid was named pTrp1. Next, pKK223-3 (Arnersham-Pharmacia) wastreated with HindIII/HincII, and the resulting DNA fragment containingrmB terminator was ligated with the product of treating pTrp1 withHindIII/PvuII. E. coli JM109 was then transformed with this ligationmixture, a strain was selected among ampicillin-resistant strains thathad the desired plasmid, and that plasmid was named pTrp2. Next, the trppromoter region was amplified by PCR with pTrp2 as a template and theoligonucleotides shown in the table as primers (combination of SEQ IDNos: 10 and 12). This DNA fragment was treated with Eco01091/NdeI andligated with the product of treating pTrp2 with Eco01091/NdeI. E. coliwas then transformed with this ligation mixture, a strain was selectedamong ampicillin-resistant strains that had the desired plasmid, andthat plasmid was named pTrp4.

TABLE 8 Primer SEQ ID No. 10 5′-sideGTATCACGAGGCCCTAGCTGTGGTGTCATGGTCGGTGATC         Eco0109 SEQ ID No. 113′-side TTCGGGGATTCCATATGATACCCTTTTTACGTGAACTTGC             NdeISEQ ID No. 12 3′-side GGGGGGGGCATATGCGACCTCCTTATTACGTGAACTTG         NdeI(2) Construction of Aldolase Gene-Expressing Plasmids ptrpALD1 andptrpALD2, and Expression in E. coliA fragment amplified from a chromosomal DNA of P. taetrolens ATCC4683using the primers shown in Table 9 (SEQ ID Nos: 9 and 13) was digestedwith NdeI/HindIII to construct plasmid ptrpALD1 inserted into theNdeI/HindIII site of pTrp4. This plasmid expresses aldolase genecomposing the amino acid sequence of SEQ ID No: 3 translated from the444th ATG in the nucleotide sequence of SEQ ID No: 1 as the initiationcodon. Furthermore, a fragment amplified from a chromosomal DNA of P.taetrolens ATCC4683 with primers (SEQ ID Nos: 9 and 14) was digestedwith NdeI/HindIII to construct plasmid ptrpALD2 inserted into theNdeI/HindIII site of pTrp4. This plasmid expresses aldolase genecomposing the amino acid sequence of SEQ ID No: 2 translated from the456th ATG in the nucleotide sequence of SEQ ID No: 1 as the initiationcodon. Each of the constructed expression plasmids was introduced intoE. coli JM109, and the transformants were cultured at 37° C. overnightin LB medium containing 50 μg/ml of ampicillin (pre-cultivation). Thepre-culture was inoculated at 1% in 50 ml of LB medium followed bycultivation at 37° C. The microbial cells were harvested, washed andsuspended in 1 ml of 20 mM Tris-HCl (pH 7.6) followed by disrupting themicrobial cells using a Multi-Beads Shocker (Yasui Kikai Corporation).The crude extract was then centrifuged at 15000 rpm for 10 minutes andthe resulting supernatant was used as a crude enzyme solution.

TABLE 9 Primer SEQ ID No. 9 ALD-3′ Hind(5′-CCG AAG CTT TCA GTTCGC CAG GCC AGC C-3′) SEQ ID No. 13 ALD-5′ Nde-1(5′-GGT TCA GTC ACA TATGGA GGT CGC TAT GTC-3′) SEQ ID No. 14 ALD-5′Nde-2(5′-ATG GAG GTC CAT TAG TCA TTG CCC GGT TCA CGC-3′)

Measuring aldolase activity using the crude enzyme and PHOG for thesubstrate, in contrast to aldolase activity for PHOG not being detectedin the E. coli harboring pTrp4 (control), PHOG-aldolase activity of 16.1U/mg protein was detected in the strain harboring ptrpALD1, whilePHOG-aldolase activity of 36.0 U/mg protein was detected in the strainharboring ptrpALD2. As a result, regardless of which aldolase comprisingthe amino acid sequence of SEQ ID No: 2 or 3 was used, both aldolaseswere demonstrated to have aldolase activity.

Example 3 Cloning of IHOG-Aldolase Derived from Pseudomonascoronafaciens Strain AJ2791

(1) Preparation of Chromosomal DNA

P. coronafaciens strain AJ2791 was cultivated at 30° C. overnight using50 ml of bouillon medium (pre-cultivation). 5 ml of this culture wasinoculated into 50 ml of bouillon medium. After the cultivation untilthe late logarithmic growth phase, 50 ml of culture was centrifuged(12000×g, 4° C., 15 minutes) to recover the microorganisms. Achromosomal DNA was then prepared according to established methods usingthese microbial cells.

(2) Cloning of Full Length Gene by Southern Analysis and ColonyHybridization

Cloning of IHOG-aldolase gene derived from P. coronafaciens strainAJ2791 (hereinafter, “PcALD”) was performed by Southern hybridizationanalysis and colony hybridization with IHOG-aldolase gene derived fromP. taetrolens strain ATCC4683 as a probe. The full length ofIHOG-aldolase gene was amplified by PCR from a chromosomal DNA of P.taetrolens ATCC4683 using the primers ALD-5′ Nde-1 (SEQ ID No: 13) andALD-3′ Hind (SEQ ID No: 9) shown in Table 9. The full length gene wasthen obtained by Southern analysis and colony hybridization with theamplified DNA fragment as a probe. The DNA probe was produced by usingDIG High Prime (Roche Diagnostics K.K.), O/N incubating at 37° C.according to the instruction manual to label the probe. Southernhybridization analysis was performed according to the manual bycompletely digesting 1 μg of chromosomal DNA of P. coronafaciens strainAJ2791 with each type of restriction enzyme, electrophoresing in 0.8%agarose gel and blotting onto a nylon membrane. Hybridization wasperformed using DIG Easy Hyb (Roche Diagnostics K.K.) by pre-hybridizingat 37° C. for 1 hour, adding the probe and then hybridizing with O/N at37° C. The membrane was washed twice at room temperature for 5 minutesusing 2×SSC, and then washed twice at 37° C. for 15 minutes using0.1×SSC. Bands were detected using the DIG Nucleotide Detection Kit. Asa result, an approximately 2.2 kbp PstI/HindIII fragment was detectedthat hybridized with the probe. Next, this PstI/HindIII fragment wasobtained by colony hybridization. 20 μg of chromosomal DNA was treatedwith PstI/HindIII followed by applying to agarose gel electrophoresisand recovering a fragment of which size was about 2.2 kbp. This was thenligated into pUC118 treated with PstI/HindIII to produce a library in E.coli JM109. The colonies were then transferred to a Nylon membranefilter (Hybond-N, Amersham) followed by alkaline denaturation,neutralization and immobilization treatment. Hybridization was performedusing DIG Easy Hyb. The Nylon membrane fitter was immersed in a bufferand pre-hybridized at 37° C. for 1 hour. Subsequently, the previouslyprepared labeled probe was added and hybridized at 37° C. for 16 hours.The Nylon membrane filter was washed twice at room temperature for 5minute using 2×SSC and then washed twice at 37° C. for 15 minutes using0.1×SSC. Colonies that hybridized with the probe were detected using theDIG Nucleotide Detection Kit (Roche Diagnostics K.K.). As a result, aclone was obtained that hybridized with the probe.

When the nucleotide sequence of plasmid DNA recovered from the positiveclone was determined, a 744 bp orf was found, and the full length of thedesired aldolase was obtained. The constructed plasmid inserted with thePcALD gene was named pUCPcALD.

E. coli JM109 was transformed with pUCPcALD, and the transformant wascultured at 37° C. for overnight in LB medium containing 50 μg/ml ofampicillin (LB-amp medium) (pre-cultivation). Thus obtained preculturewas inoculated at 1% in 50 ml of fresh LBmedium, followed by cultivationat 37° C. IPTG was added to a final concentration of 1 mM at about 2hours after the start of cultivating followed by additionallycultivating for 3 hours. After cultivating, the microorganisms wererecovered, washed, and then suspended in 1 ml of 20 mM Tris-HCl (pH 7.6)followed by disrupting the microbial cells with a Multi-Beads Shocker(Yasui Kikai Corporation). The crude extract was then centrifuged at15000 rpm for 10 minutes, and the resulting supernatant was used as acrude enzyme.

When aldolase activity was measured using the crude enzyme and PHOG as asubstrate, in contrast to aldolase activity for PHOG not being detectedin the E. coli harboring pUC18 (control), PHOG-aldolase activity of 39.3U/mg protein was detected in the strain harboring pUCADL. As a result,the gene was demonstrated to encode the desired aldolase.

Example 4 Purification of Recombinant Enzyme of Aldolase Derived fromPseudomonas taetrolens Strain ATCC4683

Recombinant aldolase derived from P. taetrolens ATCC4683 (PtALD) waspurified as described below from the soluble fraction of E. coli thathighly expresses PtALD. Aldolase activity was determined by measuringaldolase activity when using PHOG as a substrate under the followingconditions.

Reaction conditions: 50 mM Tris-HCl (pH 8.0), 2 mM PHOG, 0.2 mM NAD, 0.2mM KPi, 1 mM MgCl₂, 16 U/ml of lactate dehydrogenase and 3 μL enzyme/600μl reaction mixture, optical absorbance at 340 nm measured at 30° C.

(1) Preparation of Soluble Fraction

A loopful of E. coli JM109/ptrpALD2 cultured at 37° C. for 16 hours onLB-amp plate medium were picked from the plate, inoculated into a testtube containing 3 ml of LB-amp medium and shake-culture was performed at37° C. for 16 hours. 0.5 ml of this culture was then inoculated into ten500 ml volumetric flasks containing 50 ml of LB-amp medium followed byculturing at 37° C. for 16 hours. The microorganisms were recovered fromthe resulting culture by centrifugation, and after washing by suspendingin buffer A (20 mM Hepes-KOH (pH 7.6)), were again recovered bycentrifugation. The resulting washed microbial cells were suspended in25 ml of buffer A and then ultrasonically disrupted at 4° C. for 30minutes. The microbial cell residue was removed from the crude extractby centrifugation (×8000 rpm, 10 min×2 times) after which the resultingsupernatant was used as the crude fraction.

(2) Anionic Exchange Chromatography: Q-Sepharose FF

23 ml of the crude fraction was applied to a Q-Sepharose FF 26/10anionic exchange chromatography column (Pharmacia, CV=20 ml)equilibrated with buffer A and adsorbed onto the carrier. After washingout any unbound proteins using buffer A, the PtALD was eluted whilelinearly changing the KCl concentration from 0 M to 0.7 M (total: 140ml). The aldolase activity for PHOG was observed in the fractionequivalent to about 0.5 M.

(3) Hydrophobic Chromatography: Phenyl Sepharose HP HR 16/10

The fraction detected aldolase activity was dialyzed at 4° C. overnightagainst buffer B (20 mM Hepes-KOH (pH 7.6), 1 M ammonium sulfate, pH7.6) and then filtering the supernatant obtained with a 0.45 μm filter.The resulting filtrate was applied to a hydrophobic chromatographycolumn, Phenyl Sepharose HP HR 16/10 (Pharmacia) equilibrated withbuffer B. This procedure resulted in the aldolase being adsorbed to thecarrier.

After washing out any unbound proteins using buffer B, aldolase waseluted using liner gradient of the ammonium sulfate concentration from 1M to 0 M. The aldolase activity was detected at the elution positionwhere the concentration of ammonium sulfate was about 0.2 M.

When the fraction purified according to the chromatography proceduresdescribed above was applied to SDS-PAGE, a single band was observed atthe location corresponding to about 25 kDa with CBB staining. Theresulting recombinant PtALD solution was dialyzed at 4° C. overnightagainst buffer A. As a result of the procedure, 17 ml of 350 U/ml PtALDsolution was obtained.

Example 5 Purification of Recombinant Enzyme of Aldolase Derived fromPseudomonas coronafaciens Strain AJ2791

Recombinant aldolase derived from P. coronafaciens AJ2791 (PcALD) waspurified from the soluble fraction of E. coli that highly expressesPcALD.

A loopful of E. coli JM109/pUCPcALD cells cultivated at 37° C. for 16hours on LB-amp plate medium was picked from the plate, inoculated intoa test tube containing 3 ml of LB-amp medium and shake-culture wasperformed at 37° C. for 16 hours. 0.5 ml of this culture was theninoculated into seven pieces of 500 ml volumetric flasks containing 50ml of LB-amp medium (+0.1 mM IPTG) followed by shake-culturing at 37° C.for 16 hours. The microorganisms were recovered from the resultingculture by centrifugation, and after washing by suspending in buffer A(20 mM Hepes-KOH (pH 7.6)), were again recovered by centrifugation. Theresulting washed microbial cells were suspended in 30 ml of buffer A andthen ultrasonically disrupted at 4° C. for 30 minutes. The microbialcell residue was removed from the crude extract by centrifugation (×8000rpm, 10 min×2 times) after which the resulting supernatant was used asthe crude extraction fraction.

The resulting crude extraction fraction was purified by chromatographyusing the same procedure as the purification of recombinant PtALDdescribed in Example 4. When a fraction purified by a two-stagechromatography procedure using Q-Sepharose and Phenyl Superose wasapplied to SDS-PAGE, a single band was observed at the locationcorresponding to about 25 kDa with CBB staining. The resultingrecombinant PcALD solution was dialyzed at 4° C. overnight againstbuffer A. As a result of the aforementioned procedure, 10 ml of 108 U/mlPcALD solution was obtained.

Example 6 Synthesis of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaratefrom Indole-3-pyruvic Acid and Pyruvic Acid Using PtALD and PcALD

4-(Indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG) was synthesizedfrom indole-3-pyruvic acid and pyruvic acid using the PtALD and PcALDproduced in Examples 4 and 5 as enzyme sources. The amount of IHOG wasdetermined by HPLC analysis using the “Inertsil ODS-2” (GL SciencesInc., 5 μm, 4.6×250 mm). The analysis conditions are as indicated below.

Mobile phase: 40% (v/v) acetonitrile/5 mM tetrabutyl

ammonium dihydrogen phosphate solution

Flow rate: 1 ml/min

Column temperature: 40° C.

Detection: UV 210 nm

PtALD and PcALD were added at 1.8 U/ml and 0.8 U/ml, respectively, to areaction solution comprised of 100 mM Hepes-KOH (pH 8.5), 50 mMindole-3-pyruvic acid, 250 mM pyruvic acid and 1 mM to 5 mM MgCl₂, andallowed to react at 33° C. for 4 hours. The enzyme reaction solution wassuitably diluted and the amount of IHOG formed was quantified. As aresult, IHOG was able to be formed from the aldolases.

TABLE 10 MgCl₂ (mM) IHOG (mM) PcALD 1 4.9 5 5.4 PtALD 1 11.4 5 10.5

Example 7 Enzymatic Synthesis of Various Substituted α-Keto Acids UsingPtALD

The aldol condensation activity of pyruvic acid was measured for thevarious substrates shown in Table 11 using the PtALD produced in Example4. A reaction solution was prepared composed of 100 mM glycine-NaOH (pH9), 50 mM substrate, 250 mM sodium pyruvate, 1 mM MgCl₂ and 0.1 mg/mlPtALD, and incubated at 33° C. for 2 hours. The resulting reactionsolution was subjected to ultrafiltration with an ultrafiltrationmembrane, Microcon 10 (Amicon) for a fraction molecular weight of 10000.2 μl of the filtrate was diluted in 200 μl of 50% acetonitrile/0.1%aqueous ammonia solution followed by ESI-MS analysis.

As a result, peaks in the corresponding m/z values were detected for thesubstrates shown in Table 12, thus indicating that the desired pyruvicacid aldol condensates were formed.

TABLE 11 Aldol Condensates of Pyruvic Acid for Various Substrates UsingPtALD Exact MS [M − H]⁻ Substrate Substrate Aldol Condensate(calculated) (measured) indol-3-pyruvic acid

291.1 290.2 β-hydroxypyruvic acid

192.03 191.19 2-ketobutyric acid

190.05 189.19 3-methyl-2-oxo butanoic acid

204.06 203.08 α-ketogrutarate

234.04 233.45 pyruvic acid

176.03 175.2 D-arabinose

238.07 237.15 L-mannose

268.08 267.72

TABLE 12 Aldol Condensates of Pyruvic Acid for Various Substrates UsingPtALD Exact MS [M − H]⁻ Substrate Substrate Aldol Condensate(calculated) (measured) L-ribose

238.07 237.05 N-acetyl-L-mannosa mine

309.27 308.48

Example 8 Enzymatic Properties of PtALD

A study was conducted on the following parameters regarding the PtALDprepared in Example 4.

Basic reaction conditions: 50 mM Hepes-KOH (pH 8.0), 2 mM PHOG, 5 mMMgCl₂, 3 mM KPB (pH 8), 16 U/ml of lactate dehydrogenase, measure aoptical absorbance at 340 nm measured at 30° C.

(1) Kinetics Constants When Using PHOG as Substrate

Vmax (for PHOG)=91.7 μmol/min/mg, Km (for PHOG)=0.10 mM, Km (forMgCl₂)=0.019 mM, and Ka (for KPi)=0.95 mM were respectively determinedfrom the results shown in FIGS. 2 to 4. The aldolase activity increasedby 2.7 times as compared with a non-addition lot following addition of 1mM MgCl₂, and increased 2 times as compared with a non-addition lotfollowing addition of 5 mM KPB.

(2) pH Stability

pH stability was measured over a range of pH 3 to pH 11. The buffersused for measurement were as follows: sodium acetate buffer (pH 3, 4, 5,6), potassium phosphate buffer (pH 6, 6.5, 7, 7.5), Tris-HCl buffer (pH7, 7.5, 8, 8.5), glycine-NaOH buffer (pH 9, 9.5, 10), CAPS-NaOH buffer(pH 10, 10.5, 11). The residual activity after incubating PtALD at 37°C. for 30 minutes in each of the buffer solutions at a concentration of100 mM was measured under the basic reaction conditions. Those resultsare shown in FIG. 5.

(3) Temperature Stability

Residual activity was measured under the basic measurement conditionsafter incubating PtALD at 25 to 70° C. for 30 minutes in 100 mMpotassium phosphate buffer (pH 7.0) and 100 mM glycine-NaOH buffer (pH9.0). Those results are shown in FIG. 6.

Example 9 Synthesis of Substituted α-Keto Acid Using PtALD

The aldol condensation activity of the PtALD prepared in Example 4 wasmeasured using acetaldehyde and α-ketobutyrate. A reaction solutioncomposed of 100 mM glycine-NaOH (pH 9), 50 mM acetoaldehyde, 250 mMα-ketobutyrate, 1 mM MgCl₂ and 0.1 mg/ml RAW was prepared, and it wasincubated at 33° C. for 16 hours. The resulting reaction solution wassubjected to ultrafiltration with an ultrafiltration membrane, Microcon10 (Amicon) for a fraction molecular weight of 10000. 10 μl of thefiltrate was diluted in 200 μl of 50% acetonitrile/0.1% aqueous ammoniasolution followed by ESI-MS analysis.

As a result, in contrast to the exact mass (calculated value) of thepredicted product being 146.06, a peak (m/z=+146.8) of the m/z valuecorresponding to [M+H] was detected, thereby confirming formation of thedesired aldol condensate according to the reaction shown below.

Reference Example 1 Synthesis of4-hydroxy-4-(3-Indolylmethyl)-2-ketoglutarate (IHOG)

7.50 g (35.8 mmol, content 97.0% by weight) of indole-3-pyruvic acid and14.18 g (107.4 mmol) of oxaloacetic acid were added to 64.45 ml ofaqueous solution including 18.91 g (286.5 mmol, content: 85% by weight)of potassium hydroxide and dissolved. This mixed solution was thenstirred at 35° C. for 24 hours.

Furthermore, the mixed solution was neutralized (pH 7.0) by addition of40.0 ml of 3 N hydrochloric acid to obtain 153.5 g of a neutralizedreaction solution. This neutralized reaction solution contained 5.55 gof 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutarate, and the yield was53.3% (with respect to indole pyruvic acid).

Water was added to this neutralized reaction solution to bring to avolume of 168 ml and then passed through a resin column (diameter 4.8cm) filled with 840 ml of a synthetic adsorbent (DIAION-SP207,Mitsubishi Chemical Corporation). Furthermore, water was passed throughthe column at a flow rate of 23.5 ml/min and by recovering the productfrom 1.73 to 2.55 (L/L-R), an aqueous solution containing 3.04 g ofhighly pure 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutarate was obtainedat a yield of 54.7% (with respect to the amount charged into the resin).

(NMR Measurement)

¹H-NMR (400 MHz, D₂O): 3.03 (d, 1H, J=14.6 Hz), 3.11 (d, 1H, J=14.6 Hz),3.21 (d, 1H, J=18.1 Hz), 3.40 (d, 1H, J=18.1 Hz), 7.06-7.15 (m, 3H),7.39 (d, 1H, J=7.8 Hz), 7.66 (d, 1H, J=7.8 Hz)

¹³C-NMR (100 MHz, D₂O): 35.43, 47.91, 77.28, 109.49, 112.05, 119.44,119.67, 121.91, 125.42, 128.41, 136.21, 169.78, 181.43, 203.58

Reference Example 2 Synthesis of4-phenylmethyl-4-hydroxy-2-ketoglutarate (PHOG)

5.0 g (30.5 mmol) of phenylpyruvic acid and 12.1 g (91.4 mmol) ofoxaloacetic acid were added to 25 ml of water in which 13.8 g ofpotassium hydroxide (purity: 85%) was dissolved and the resultingmixture was allowed to react at room temperature for 72 hours. The pHvalue of this reaction solution was then adjusted to 2.2 usingconcentrated hydrochloric acid followed by extracting with ethylacetate. After washing the organic layer with a saturated aqueous NaClsolution and drying with anhydrous magnesium sulfate and thenconcentrating, the residue was obtained. The residue was recrystallizedfrom ethyl acetate and toluene to obtain 2.8 g (11.3 mmol) of4-phenylmethyl-4-hydroxy-2-ketoglutarate in the form of crystals.

(NMR Measurement)

¹H-NMR (D₂O) δ: 2.48 (d, J=14.4 Hz, 0.18H), 2.60 (d, J=14.4 Hz, 0.18H),2.85-3.30 (m, 3.64H), 7.17-7.36 (m, 5H)

(Measurement of Molecular Weight)

ESI-MS Calculated Value for C₁₂H₁₂O₆=252.23, analytical value=251.22(MH⁻)

INDUSTRIAL APPLICABILITY

The aldolase of the present invention is a novel aldolase that catalyzesan aldol condensation reaction of indole pyruvic acid and pyruvic acid(or oxaloacetic acid), and may be preferably used to synthesize4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate (IHOG), which is useful asan intermediate in monatin synthesis.

1. A process for producing a substituted a-keto acid of formula (3):

wherein R¹ represents a hydrogen atom, an unsubstituted alkyl grouphaving 1 to 8 carbon atoms, an alkyl group having 1 to 8 carbon atomscomprising at least one substituent, an unsubstituted alkoxyl grouphaving 1 to 8 carbon atoms, an alkoxyl group having 1 to 8 carbon atomscomprising at least one substituent, an unsubstituted carboxyalkyl grouphaving 2 to 9 carbon atoms, a carboxyalkyl group having 2 to 9 carbonatoms comprising at least one substituent, an unsubstituted aryl havingup to 20 carbon atoms, an aryl having up to 20 carbon atoms comprisingat least one substituent, an unsubstituted aralkyl group having up to 20carbon atoms, an aralkyl group having up to 20 carbon atoms comprisingat least one substituent, an unsubstituted heterocycle-containinghydrocarbon group having up to 11 carbon atoms, a heterocycle-containinghydrocarbon group having up to 11 carbon atoms comprising at least onesubstituent, a hydroxyl group or an ester derivative thereof, whereinsaid substituent of R¹ is selected from the group consisting of ahalogen atom, a hydroxyl group, an alkyl group having up to 3 carbonatoms, an alkoxy group having up to 3 carbon atoms, and an amino group;and R² represents a hydrogen atom, an unsubstituted alkyl group having 1to 8 carbon atoms, an alkyl group having 1 to 8 carbon atoms comprisingat least one substituent, an unsubstituted alkoxyl group having 1 to 8carbon atoms, an alkoxyl group having 1 to 8 carbon atoms comprising atleast one substituent, an unsubstituted carboxyalkyl group having 2 to 9carbon atoms, a carboxyalkyl group having 2 to 9 carbon atoms comprisingat least one substituent, an unsubstituted aryl having up to 20 carbonatoms, an aryl having up to 20 carbon atoms comprising at least onesubstituent, an unsubstituted aralkyl group having up to 20 carbonatoms, an aralkyl group having up to 20 carbon atoms comprising at leastone substituent, an unsubstituted heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms comprising at least one substituent,a hydroxyl group or an ester derivative thereof, wherein saidsubstituent of R² is selected from the group consisting of a halogenatom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, analkoxy group having up to 3 carbon atoms and an amino group; and when R¹represents a hydrogen atom, a methyl group or a carboxymethyl group informula (1), R² does not represent a hydrogen atom; comprising areaction of the substituted α-keto acid of formula (1):

wherein R¹ has the same meaning as R¹ in formula (3), with thesubstituted α-keto acid of formula (2):

wherein R² has the same meaning as R² in formula (3); wherein thereaction is catalyzed by a protein that has aldolase activity and isselected from the group consisting of: a protein comprising the aminoacid sequence of SEQ ID No: 16; and a protein having the amino acidsequence of SEQ ID No: 16 except that it contains substitution,deletion, insertion, or addition of one to ten amino acid residues inthe amino acid sequence of SEQ ID No:
 16. 2. The process according toclaim 1, wherein R² is a hydrogen atom or a carboxyl group.
 3. Theprocess according to claim 2, wherein R² is a hydrogen atom.
 4. Theprocess according to claim 1, wherein R¹ is a benzyl group or a3-indolylmethyl group, and R² is a hydrogen atom or a carboxyl group. 5.The process according to claim 1, wherein said protein is a protein thatcomprises the amino acid sequence of SEQ ID NO:
 16. 6. The processaccording to claim 1, wherein said protein is a protein that has theamino acid sequence of SEQ ID No: 16 except that it containssubstitution, deletion, insertion, or addition of one to ten amino acidresidues in the amino acid sequence of SEQ ID No:
 16. 7. A process forproducing a substituted α-keto acid of formula (3′):

wherein R¹ represents a hydrogen atom, an unsubstituted alkyl grouphaving 1 to 8 carbon atoms, an alkyl group having 1 to 8 carbon atomscomprising at least one substituent, an unsubstituted alkoxyl grouphaving 1 to 8 carbon atoms, an alkoxyl group having 1 to 8 carbon atomscomprising at least one substituent, an unsubstituted carboxyalkyl grouphaving 2 to 9 carbon atoms, a carboxyalkyl group having 2 to 9 carbonatoms comprising at least one substituent, an unsubstituted aryl havingup to 20 carbon atoms, an aryl having up to 20 carbon atoms comprisingat least one substituent, an unsubstituted aralkyl group having up to 20carbon atoms, an aralkyl group having up to 20 carbon atoms comprisingat least one substituent, an unsubstituted heterocycle-containinghydrocarbon group having up to 11 carbon atoms, a heterocycle-containinghydrocarbon group having up to 11 carbon atoms comprising at least onesubstituent, a hydroxyl group or an ester derivative thereof, whereinsaid substituent of R¹ is selected from the group consisting of ahalogen atom, a hydroxyl group, an alkyl group having up to 3 carbonatoms, an alkoxy group having up to 3 carbon atoms, and an amino group;R³ represents a hydrogen atom or a carboxyl group; and R⁴ represents ahydrogen atom, an unsubstituted alkyl group having 1 to 8 carbon atoms,an alkyl group having 1 to 8 carbon atoms comprising at least onesubstituent, an unsubstituted alkoxyl group having 1 to 8 carbon atoms,an alkoxyl group having 1 to 8 carbon atoms comprising at least onesubstituent, an unsubstituted carboxyalkyl group having 2 to 9 carbonatoms, a carboxyalkyl group having 2 to 9 carbon atoms comprising atleast one substituent, an unsubstituted aryl having up to 20 carbonatoms, an aryl having up to 20 carbon atoms comprising at least onesubstituent, an unsubstituted aralkyl group having up to 20 carbonatoms, an aralkyl group having up to 20 carbon atoms comprising at leastone substituent, an unsubstituted heterocycle-containing hydrocarbongroup having up to 11 carbon atoms, a heterocycle-containing hydrocarbongroup having up to 11 carbon atoms comprising at least one substituent,a hydroxyl group or an ester derivative thereof, wherein saidsubstituent of R⁴ is selected from the group consisting of a halogenatom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, analkoxy group having up to 3 carbon atoms, and an amino group; comprisinga reaction of the compound of formula (1′):

wherein R¹ and R³ have the same meanings as defined in formula (3′),with the substituted α-keto acid of formula (2′):

wherein R⁴ has the same meaning as defined in formula (3′); wherein thereaction is catalyzed by a protein that has aldolase activity and isselected from the group consisting of: a protein comprising the aminoacid sequence of SEQ ID No: 16; and a protein having the amino acidsequence of SEQ ID No: 16 except that it contains substitution,deletion, insertion, or addition of one to ten amino acid residues inthe amino acid sequence of SEQ ID No:
 16. 8. The process according toclaim 7, wherein R⁴ is a hydrogen atom or a carboxyl group.
 9. Theprocess according to claim 7, wherein said protein is a protein thatcomprises the amino acid sequence of SEQ ID NO:
 16. 10. The processaccording to claim 7, wherein said protein is a protein that has theamino acid sequence of SEQ ID No: 16 except that it containssubstitution, deletion, insertion, or addition of one to ten amino acidresidues in the amino acid sequence of SEQ ID No:
 16. 11. A process forproducing a substituted α-keto acid of formula (4):

from oxaloacetic acid or pyruvic acid, and indole-3-pyruvic acid whereinthe reaction is catalyzed by a protein that has aldolase activity and isselected from the group consisting of: a protein comprising the aminoacid sequence of SEQ ID No: 16; and a protein having the amino acidsequence of SEQ ID No: 16 except that it contains substitution,deletion, insertion, or addition of one to ten amino acid residues inthe amino acid sequence of SEQ ID No:
 16. 12. The process according toclaim 11, wherein said protein is a protein that comprises the aminoacid sequence of SEQ ID NO:
 16. 13. The process according to claim 11,wherein said protein is a protein that has the amino acid sequence ofSEQ ID No: 16 except that it contains substitution, deletion, insertion,or addition of one to ten amino acid residues in the amino acid sequenceof SEQ ID No: 16.